癌基因AGR2和双联苄化合物介导的DNA损伤信号在前列腺癌细胞衰老和凋亡中的作用
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摘要
前列腺癌(Prostate cancer, PCa)是老年男性常见的恶性肿瘤,其死亡率仅次于肺癌。数据显示,2012年新诊断的前列腺癌患者为110万,约占新增癌症病例总数的15%,其中四期PCa患者5年生存率仅为20%,10年生存率低于10%。虽然我国PCa的发病率明显低于欧美国家,但近年来随着人口老龄化、生活环境和饮食结构的改变等,我国PCa发病率增长迅速,且中晚期患者的比例远远大于国外,恶性程度高,预后差。预计2020年前后,我国前列腺癌发病率可能会超过60-70/10万。前列腺癌已成为临床关注的男性恶性疾病之一。
PCa作为激素依赖性肿瘤,去势治疗是首选标准方案,约80%的前列腺癌对去势治疗敏感。然而绝大多数PCa患者经去势治疗1-2年后,肿瘤复发,成为激素难治性前列腺癌(Hormone Refractory Prostate Cancer, HRPC),抵抗内分泌治疗,抗凋亡能力强,伴随肿瘤的侵袭转移,死亡率高,预后差。由于HRPC的发展和演变是复杂的多因素、多信号通路参与的过程,目前的研究主要侧重于两方面,一是HRPC发生发展的机制,二是新靶点的鉴定及其干预药物。我们利用生物信息学技术,通过分析癌症和肿瘤基因图谱(Cancer Genome Atlas, TCGA)计划所提供的大规模基因组测序数据,绘制了人前列腺癌的基因组变异图谱,并进行系统分析,确定与HRPC的分期、侵袭转移、药物耐受等恶性表型密切相关的基因,筛选出AGR2为候选靶基因之一。由于TCGA的数据基础主要来自欧美人群,我们又利用中国汉族人群PCa患者的临床样本进行分析,发现AGR2与中国汉族人群PCa患者的恶性表型密切相关。进一步研究发现,降低AGR2在PCa细胞中的表达,可通过DNA损伤应答机制诱导细胞衰老。同时发现具有拓扑异构酶抑制活性的天然联苄化合物,可通过DNA损伤应答信号诱导PCa细胞衰老,而在DNA损伤程度较强时诱导激素非依赖PCa细胞凋亡。另外还发现,联苄化合物可通过抑制雄激素受体的表达和功能诱导激素依赖性PCa细胞凋亡。
第一部分基于前列腺癌基因组图谱的AGR2功能分析与AGR2缺乏介导的DNA损伤诱导前列腺癌细胞衰老的机制作用
目前已知的癌症有200多种,但是所有肿瘤在特殊类别(分型)或发展的不同分期方面都发现有基因组的特异变化,而正是基因组的改变导致了细胞分化、发育和生长通路的异常,从而引发细胞增殖、生长的失控。本论文通过分析癌症和肿瘤基因图谱(Cancer Genome Atlas,TCGA)计划所提供的大规模基因组测序数据,绘制了人类前列腺癌的基因组变异图谱,并进行系统分析,旨在找到所有癌基因和抑癌基因的微小变异,分析其在前列腺癌发生、发展的变化趋势及可能的作用,确定能够驱动PCa发生的潜在drivers或治疗靶点,以期发现前列腺癌发生发展和治疗的新策略。
前梯度同源蛋白2(Anterior gradient protein2homolog, AGR2)是本课题组通过前列腺癌基因组图谱分析得到的候选靶基因之一。现已报道多种肿瘤的发生与发展伴随着AGR2表达的上调,包括前列腺癌、乳腺癌、肺癌和胰腺癌等等。在正常的前列腺组织中,AGR2受雄激素诱导表达,但是对前列腺癌样品的显微切割分析表明AGR2的mRNA和蛋白水平都较临近的良性组织有显著的升高。最近有报道指出,在前列腺癌患者的尿液中检测到高水平的AGR2,因此AGR2有望作为前列腺癌的尿检标志物。到目前为止,尚未报道过汉族人群前列腺癌组织中AGR2蛋白的表达情况及作用机制。本课题组对汉族人群前列腺组织AGR2蛋白的表达做了检测,发现在汉族人群前列腺癌组织中AGR2的表达水平显著高于良性组织,同时在前列腺癌细胞系中也观察到AGR2的表达明显高于前列腺正常上皮细胞,并首次发现和提出抑制前列腺癌细胞中AGR2的表达可导致显著性细胞衰老。通过详细剖析AGR2基因在前列腺癌细胞中的生物学功能,表明AGR2在前列腺癌检测和治疗中具有潜在价值。
一、前列腺癌基因组图谱的绘制
1.前列腺癌转录组严重失调。通过对256例前列腺癌组织以及45例前列腺癌旁正常组织的转录组测序(Whole Transcriptome Shotgun Sequencing, RNASeq)数据分析,癌组织中24%以上的基因较正常组织有显著性上调或下调,其中蛋白编码基因(Protein-coding genes, PCGene)失调率为28.53%,长链非编码RNA(long non-coding RNA,lncRNA)失调率为27.64%,假基因(Pseudogene)失调率为16.61%。
2.前列腺癌体细胞基因组中存在DNA序列拷贝数的畸变。通过对258例前列腺癌样本以及相匹配的正常对照组织的高分辨率单核苷酸多态性基因分型芯片(Affymetrix Genome-Wide Human SNP Array6.0,SNP6.0)分析,共找到了2448个拷贝数异常的基因,包括961个PCGene,808个lncRNA和679个Psuedogeneo
3.前列腺癌表观遗传学分析。通过对252例前列腺癌组织以及49例前列腺癌旁正常组织的全基因组甲基化芯片(Illumina Infinium HumanMethylation450BeadChip)分析,得到了2617个前列腺癌特异性过甲基化基因,提示前列腺癌中存在全基因组甲基化失衡。对259例前列腺癌组织以及50例前列腺癌旁正常组织的：miRNA深度测序(miRNASeq)数据分析显示,前列腺癌组织中36.90%的miRNA较正常组织有显著性上调或下调。
二、基于图谱信息和实验数据对AGR2的表达和功能的分析
1.AGR2基因在PCa组织中表达显著上调。通过对前列腺癌INASeq数据分析,发现AGR2基因在前列腺组织中有较高的基础丰度,而且在PCa组织中的表达丰度上调非常显著。而DNA序列拷贝数数据分析结果表明,AGR2的拷贝数在正常组织和癌组织中并无显著差异。另外,全基因组甲基化芯片分析结果显示,AGR2在肿瘤组织中并无异常甲基化修饰。这些结果表明,AGR2在PCa中的高表达并非拷贝数增加或甲基化异常,而mRNA稳定性增加、或在转录水平表达增强等机制可能参与其调控,尚需进行研究。
2.AGR2在中国汉族人群PCa患者的组织中表达显著上调。由于TCGA的数据和文献报道主要来自欧美国家人群,而PCa的发生与种族有关。到目前为止,中国汉族人群前列腺癌患者组织中AGR2蛋白的表达情况及功能尚未报道。本课题通过免疫组化技术分析汉族人群良性前列腺增生、前列腺癌组织中AGR2蛋白的表达情况,结果显示,汉族人群前列腺癌组织中AGR2的表达水平显著高于良性组织,与欧美人群的表达趋势一致。Western blotting结果显示,AGR2在前列腺癌细胞系中的表达明显高于前列腺非恶性上皮细胞。因此,上述结果显示,AGR2基因在前列腺癌组织和细胞中的表达调控、生物学功能都有待进一步研究,以阐明其在前列腺癌发生发展和治疗中的潜在价值。
3.AGR2在前列腺癌中的功能。基于RNASeq数据组对AGR2的连坐效应相关分析(Guilt-by-Association,GBA)提示,AGR2在前列腺癌中的功能可能与细胞周期、DNA损伤修复、细胞粘附等相关。文献已有报道,AGR2与细胞的增殖、侵袭转移等有关,但机制尚需进一步研究。
三、DNA损伤应答参与AGR2介导的前列腺癌细胞衰老的机制研究
1.下调AGR2抑制PCa细胞增殖。上述结果表明,AGR2在中国汉族人群PCa组织、细胞系中高表达,我们首先在AGR2表达相对较高的PC3细胞中通过RNAi下调AGR2的表达,观察细胞表型的变化。MTT结果显示,下调AGR2后,显著抑制PC3细胞的增殖。相反,在AGR2表达相对较低的LNCaP和DU145细胞中转染AGR2的表达质粒,细胞的存活数显著增加。BrdU掺入实验结果显示,三种PCa细胞中下调AGR2的表达后,BrdU的掺入显著降低,表明AGR2促进细胞增殖。Western blotting结果显示,下调AGR2后,抑制PCa细胞增殖、存活信号通路,如p-Akt表达水平下降,但Akt总蛋白的表达水平并无变化；p-ERK的水平略有下调,对总ERK蛋白的表达水平无影响。
2.下调AGR2阻滞PCa细胞周期进程。免疫组化结果显示,PCa组织中AGR2的高表达伴随着周期蛋白Cyclin D1的表达上调,而周期抑制因子p16、pRb在良性前列腺增生、前列腺内皮肉瘤中表达升高,但在PCa组织中表达显著下调。IINAi结果显示,下调AGR2的表达,PCa细胞显著阻滞于G1期。
3.抑制AGR2的表达诱导前列腺癌细胞衰老。利用siRNA抑制AGR2的表达后,PCa细胞扁平胀大呈现明显的细胞衰老形态。衰老相关β-半乳糖苷酶(senescence associated P-galactosidase,SA-β-Gal)染色和衰老相关异染色质聚集灶(Senescence-associated heterochromatin foci,SHAF)分析进一步确认抑制AGR2表达可诱导前列腺癌细胞衰老。
4.干扰AGR2的表达上调细胞周期抑制因子从而引起细胞衰老。在前列腺癌细胞系LNCaP(表达野生型p53)和PC3(p53无)中抑制AGR2表达后,p21以p53非依赖的方式表达上调,Cyclin D1、pRb的表达显著下降,p16的表达有所增加。RNAi实验结果表明,下调AGR2后诱导的LNCaP和PC3细胞衰老过程依赖于p21的激活。而在DU145细胞中(表达野生型PTEN,无功能性p53),下调AGR2的表达,使PTEN的表达有所增加,显著诱导p27的表达,而p16的表达有所增加,p21的变化不显著。进一步分析证实下调AGR2诱导的DU145细胞衰老则部分依赖于p27信号通路。
5.抑制AGR2的表达诱导细胞衰老伴随DNA损伤。由于DNA损伤应答信号是诱导细胞衰老的主要机制之一,我们利用荧光抗体标记、流式分析DNA损伤的标志蛋白yH2AX的变化,结果显示,下调AGR2表达后,LNCaP和PC3细胞中yH2AX的表达显著增加,DU145细胞中yH2AX表达上调,但较LNCaP和PC3细胞弱,表明,DNA损伤参与AGR2下调诱导的细胞衰老过程,但机制尚需进一步探讨。
第二部分DNA损伤应答抑制DNA损伤修复引起细胞衰老或凋亡的机制研究
由于下调癌基因AGR2的表达,可诱导PCa细胞衰老,使我们进一步分析诱导细胞衰老作为PCa治疗的可能性。由于衰老细胞缺乏增殖能力,这无疑有利于肿瘤的治疗,因此诱导肿瘤细胞衰老(Prosenescence therapy)成为近年来的研究热点。研究发现,化疗(如DNA损伤药物顺铂、多柔比星等)后,肿瘤组织中除了凋亡细胞外,也产生衰老细胞,称为TIS (Therapy-Induced Senescence)。
DNA损伤药物是目前研究较多的促进细胞衰老的诱导剂。药物诱导的DNA损伤类型有多种,例如喜树碱和依托泊苷可以通过捕获拓扑异构酶-DNA复合物触发DNA单链或双链的断裂；顺铂可与DNA结合,产生链内交联与链间交联,从而破坏DNA的功能；环磷酰胺可使DNA分子中的鸟嘌呤N7烷化后除去,留下缺失碱基后的空隙,造成移码突变。并且一种DNA损伤剂往往可以同时引起几种类型的损伤,其损伤效应的大小和类型与剂量及细胞所处的周期状态有关。
对于不同的DNA损伤病变,特异的DNA损伤检控点通路将被激活。DNA损伤检控点通路是由一系列检控蛋白构成的复杂的信号转导网络,在细胞周期转换、DNA复制和染色体分离中发挥重要的作用,并对DNA损伤做出及时应答启动修复,以防止基因突变和遗传不稳定的发生。研究表明,毛细血管扩张性共济失调症突变蛋白(ataxia-telangiectasia mutated, ATM)、毛细血管扩张性共济失调症突变Rad3相关蛋白(ATM and Rad3-related,ATR)在DNA损伤应答系统中起到了至关重要的作用。ATM主要应对双链断裂,当DNA双链受损时,ATM发生自磷酸化并激活一系列下游分子,包括Chk2, BRCA1和NBS1等,在这些因子的共同作用下,细胞的应激系统被启动,产生周期阻滞,并进行DNA修复,细胞衰老和凋亡也可能随之发生。当细胞受到严重的DNA复制压力时ATR将被激活,并磷酸化Chkl,激活下游Cdc25分子,抑制CDK1/Cyclin B复合物活性使细胞周期阻滞,进而启动DNA修复程序。DNA双链断裂主要有两种修复机制,同源重组(homologous recombination,HR)和DNA末端链接(nonhomologous end joining,NHEJ)。HR修复是利用细胞内的染色体两两对应的特性,若其中一条染色体上的DNA发生双股断裂,则另一条染色体上对应的DNA序列即可当作修复的模版来回复断裂前的序列；NHEJ修复不需要任何模版,修复蛋白Ku70/86可以直接将双股裂断的末端彼此拉近,再借由DNA黏合酶的帮助下,将断裂的两股重新接合。DNA损伤检控点信号转导通路功能和DNA修复机制异常,DNA损伤不能及时得到修复,细胞将会走向衰老或凋亡。本课题组研究发现,双联苄化合物RD,能够诱导前列腺癌细胞DNA损伤,抑制DNA损伤修复,最终诱导前列腺癌细胞走向衰老或凋亡。
一、低剂量片叶苔素D诱导前列腺癌细胞产生p21依赖的细胞衰老
1.低剂量RD诱导前列腺癌细胞衰老。0.5μmol/L的RD处理细胞10天后,前列腺癌细胞PC3、DU145和LNCaP均出现细胞克隆能力显著下降、周期阻滞、细胞形态扁平胀大等细胞衰老特征。衰老相关β-半乳糖苷酶染色和衰老相关分泌表型(enescence-associated secretory phenotype,SASP)分析证实,RD诱导前列腺癌细胞产生明显的细胞衰老。DNA损伤标志蛋白yH2AX的免疫荧光结果显示,低剂量的P.D诱导前列腺癌细胞产生了明显的DNA损伤,Western-blotting结果显示,DNA损伤检控点蛋白Chkl/2和损伤修复相关蛋白Ku70/86与p-BRCAl表达受到抑制。以上结果提示,低剂量RD可能通过诱导引起前列腺癌细胞DNA损伤并抑制损伤修复,从而诱导细胞衰老。
2.低剂量RD诱导的细胞衰老依赖于p21的激活。Western-blotting和实时定量PCR结果显示,RD处理后,前列腺癌细胞p21的表达显著增高。利用siRNA特异性的降低p21的表达,可显著逆转RD诱导的细胞衰老。
3.RD诱导荷瘤动物体内癌细胞衰老。裸鼠荷瘤实验表明,RD能显著抑制荷瘤动物体内癌细胞生长。衰老相关β-半乳糖苷酶染色和衰老相关异染色质聚集灶分析表明,RD处理后,裸鼠体内癌细胞出现明显的细胞衰老。DNA损伤标志蛋白γH2AX的免疫组化结果显示,RD诱导裸鼠体内癌细胞出现明显的DNA损伤。免疫组化结果显示p21在RD处理组的裸鼠癌细胞中的表达显著上调,提示RD诱导裸鼠体内癌细胞衰老的机制可能与p21的激活有关。
二、片叶苔素D诱导前列腺癌细胞DNA损伤并抑制损伤修复
1.片叶苔素D诱导前列腺癌细胞DNA损伤,激活损伤应答程序。细胞周期分析结果显示,终浓度10μmol/L的RD处理细胞24h后,前列腺癌细胞细胞周期被明显阻滞在G2/M期,并伴随大量的细胞凋亡。yH2AX焦点分析、微核试验和彗星实验结果均证实,10μmol/L的RD诱导前列腺癌细胞PC3产生明显的DNA损伤。时相分析显示,DNA损伤检控点通路ATM/Chk2和ATR/Chkl在处理1h后显著激活。ATM/ATR抑制剂处理细胞后,能部分逆转片RD导致的细胞增殖抑制。
2.片叶苔素D显著抑制DNA损伤修复。Western-blotting结果显示10μmol/L的RD显著抑制DNA损伤修复相关蛋白BRCA1的表达；DNA修复活性检测表明,RD显著抑制了HR和NHEJ修复系统功能,并抑制了DNA修复酶Ku70/86的表达和活性。基因芯片结果分析显示,受RD影响的基因主要集中在细胞增殖,细胞周期,DNA损伤和修复,以及凋亡相关通路。
3.片叶苔素D诱导荷瘤动物癌细胞DNA损伤。裸鼠荷瘤实验表明,RD能显著抑制荷瘤动物体内癌细胞生长。Western-blotting和免疫荧光结果显示,RD能够改裸鼠体内癌细胞DNA损伤修复以及细胞凋亡相关通路基因的表达。其中DNA损伤标志蛋白yH2AX的显著上调表明,RD处理的裸鼠癌细胞存在明显的DNA损伤。
第三部分双联苄化合物抑制雄激素依赖性前列腺癌细胞增殖机制研究
随着肿瘤细胞生物学和肿瘤分子生物学研究的发展,人类对癌症的认识越来越深入,在肿瘤治疗方面取得了很多振奋人心的成果。例如：小分子化合物多西紫杉醇对去势治疗失败的转移性前列腺癌患者显示出明显生存优势,极大的改善了晚期前列腺癌患者的生存期。然而过半的前列腺癌患者存在药物不敏感及耐药现象,因而亟待研发新的抗肿瘤药物。双联苄(Bisbibenzyls)是从苔藓植物分离提纯得到的一类大环多酚类化合物,这类化合物结构新颖、多变,联苄之间具有连接方式和取代基团多样化的特点。前期研究发现这种化合物具有抗肿瘤、抗炎、抗真菌等多种生物活性,具有良好的开发应用潜力和前景。本课题组基于双联苄类化合物片叶苔素C(Riccardin C)、地钱素M(Marchantin M,MM)和片叶苔素D(Riccardin D)对雄激素依赖性细胞系LNCaP的增殖抑制作用,研究了其具体机制。
近年来研究显示,P13K信号途径对雄激素非依赖性前列腺癌的发生起重要作用,而AR信号通路则是激素依赖性前列腺癌增殖的关键途径。雄激素受体(Androgenreceptor,AR)是一种核受体型转录因子,当雄激素与AR结合后,会引起AR构象的改变,使AR与热休克蛋白90(HSP-90)解离,随即活化而转移至细胞核内,并形成同源二聚体与雄激素受体应答元件(AREs)结合,诱导下游靶基因的转录表达,促进细胞增殖。因此,有效抑制雄激素依赖的前列腺癌中AR的表达和功能一直是倍受关注的热点话题。本课题研究结果显示,双联苄化合物片叶苔素C(Riccardin C)、MM和RD能显著抑制雄激素依赖的前列腺癌细胞LNCaP的增殖能力。由于AR是LNCaP细胞增殖存活的关键调控因子,我们研究了双联苄化合物对AR表达和功能的影响以及与LNCaP增殖抑制之间的关系。
一、双联苄化合物抑制AR的表达和功能促进LNCaP细胞凋亡
1.RC、MM和RD抑制雄激素依赖的前列腺癌细胞LNCaP恶性增殖。RC、MM和RD将LNCaP细胞周期阻滞在G0/G1期,并伴随着p53和p21蛋白水平的增高；Western-blotting结果显示,双联苄化合物处理后Bax/Bc12蛋白比例和PARP剪切形式明显增加,表明LNCaP细胞产生明显的凋亡。
2. LNCaP细胞凋亡伴随着AR表达和活性的降低。荧光素酶报告基因分析和Western-blotting结果显示,RC、MM和RD明显抑制LNCaP细胞AR的转录活性和蛋白表达。当双联苄化合物处理后,LNCaP细胞中PSA启动子活性降低,免疫共沉淀(co-IP)结果显示AR与SRC-1结合减弱,提示AR的功能受到明显抑制。
3.双联苄化合物抑制蛋白酶体活性诱导LNCaP细胞自噬。RC、MM或RD处理细胞24h后,蛋白酶体活性显著下降,Western-blotting和co-IP结果显示,泛素化AR在细胞内蓄积。LC3蛋白表达及免疫荧光分析证实双联苄化合物引起LNCaP细胞自噬,AR、 LC3及Ub的公定位分析提示,泛素化AR的降解与细胞自噬有关。
第四部分结论及创新点
一、结论
1.前列腺癌组织基因组较正常组织出现了明显的变异。
2.AGR2在前列腺组织中高表达,抑制AGR2诱导前列腺癌细胞衰老。
4.片叶苔素D诱导前列腺癌细胞DNA损伤并抑制损伤修复,引起前列腺癌细胞衰老或凋亡。
5.双联苄化合物能够抑制激素依赖的前列腺癌细胞AR的表达和功能,从而抑制其恶性增殖。
二、创新点
1.利用大样本、多平台、高通量的基因组学手段,细致绘制了前列腺癌基因图谱。为研究前列腺癌细胞发生、发展机制,探索新的诊断和治疗方法,提供了坚实的理论基础。
2.首次报道AGR2在汉族人群前列腺癌患者中的表达分布,首次发现和提出在前列腺癌细胞中抑制AGR2的表达可诱导癌细胞衰老。
2.首次报道双联苄化合物抑制AR的表达和功能,进而促进细胞凋亡。由于LNCaP细胞表达的是突变型AR,双联苄化合物的这种抑制作用将有益于AR持续活化的前列腺癌的治疗。
3.首次报道片叶苔素D可诱导前列腺癌细胞DNA损伤并抑制损伤修复,从而导致癌细胞衰老或凋亡。由于DNA损伤修复机制是大多数DNA损伤类化疗药物敏感性的关键因素之一,片叶苔素D不仅能诱导前列腺癌细胞DNA损伤,并能进一步抑制损伤的修复,这些特点将使片叶苔素D成为很有潜力和竞争力的抗肿瘤候选药物。
Rates of detection of prostate cancer (PCa) vary widely across the world, and PCa is the most common cancer in men in the Western countries. While improved early detection significantly decreased mortality, PCa still remains the second leading cause of cancer-related death in Western men. Although the incidence of prostate cancer was lower than the Western countries', in recent years confirmed PCa, especially for advanced or metastatic PCa, represents an obviously rising trend in China with the aging and the diet habit changing.By2020, the incidence of PCa in China is rising to60-70/100,000. PCa has become one of the threats to men's health in China.
Features such as a high prevalence rate, a long latency period, and complicated pathogenesis have made PCa a desirable tumor to treat. Initially, PCa is androgen-dependent which depends largely on androgen receptor (AR) signaling for growth and maintenance. Androgen deprivation therapy has become the standard first-line treatment for hormone naive PCa, and approximately80%of patients undergo a "flare" response. Unfortunately, the development of hormone-refractory prostate cancer (HRPC) which associated with poor prognosis, high apoptosis-resistance and high mortality will be observed to occur within one or two years after androgen deprivation therapy. There are few efficacious treatment options available for curing, or even improving the survival and quality of life in patients with HRPC. Complicated factors and signals involves in the development of HRPC. Nevertheless, the underlie mechanism of PCa is still remains elusive. Therefore, novel strategies targeting the molecular basis of PCa progression and producing synergies of different signals are highly desirable.We present a large-scale, multi-omic interrogation on the alterations of genes in TCGA datasets, and identified a list of genetic disorders which is associated with malignant phenotypes such as invasion, metastasis, and drug resistance. We then took advantage of the profiles of Chinese Han PCa samples, and found AGR2overexpression was associated with the malignant phenotypes in Chinese Han PCa patients.Further study showed knockdown of AGR2caused DNA damage and resulted incellular senescence in PCa cells.Meanwhile we found natural product bisbibenzyls triggered DNA damage, leading to cellular senescence (at low concentration) and apoptotic cell death (at high concentration), which contributed to its antitumor effect in PCa cells.
Part I Analysis of functional modules of AGR2based on PCa genomic data, and AGR2i-induced DNA damage is involved in regulation of cellular senescence
PCa is the second most common cancer diagnosed in men worldwide. The prevalence of prostate cancer is growing, with an estimated1.1million men worldwide diagnosed in2012, accounting for15percent of the cancers diagnosed in men. In Stage IV prostate cancer, the5-year survival rate is about20%, and the10-year survival is above10%.There are more than200different types of cancer. It has been shown genome-wide genetic changes are correlated with specific types of cancer or the stages of tumor development. It is characterized by a progression of these changes at the cellular, genetic and epigenetic level that ultimately reprogram a cell to undergo uncontrolled cell division, thus forming a malignant mass. The Cancer Genome Atlas (TCGA) is a project to catalogue genetic mutations responsible for cancer, using genome sequencing and bioinformatics. We represented an effort in the War on Cancer that is applying high-throughput genome analysis techniques based on TCGA data to improve our ability to diagnose, treat, and prevent cancer through a better understanding of the genetic basis of this disease.
Anterior gradient protein2homolog (AGR2) is one of candidate therapeutic target genes in PCa arising from the analysis of TCGA data. Current studies showed elevated levels of AGR2in adenocarcinomas of the prostate, breast, lung and pancreas. AGR2is an androgen-inducible protein expressed in normal prostate tissue, and its expression is significantly elevated in PCa tissue compared to benign prostatic epithelial tissue. Urine sediment tests determined AGR2transcript levels to be elevated, and AGR2has been proposed as one of the potential markers to detect malignant cells in the voided urine from PCa patients. In this study, we first observed that AGR2was overexpressed in Chinese Han PCa tissues, and AGR2expression profiles varied among cell lines, with PC3cells being the highest level, LNCaP and DU145relatively less, and hardly detectable in human normal prostate epithelial cell line RWPE1. Here, a potential role of AGR2in cellular senescence was first investigated as well.
一、The genomic landscape of prostate cancer
1. Dysregulation of transcriptome in PCa.
To get a comprehensive overview on transcriptome in PCa, we analyzed RNA sequencing (RNAseq) profiles of256human tumor specimens and45normal specimens from TCGA. We identified24%genes that were up-regulated or down-regulated in the tumor specimen, including28.53%protein-coding gene (PCGene),27.64%long non-coding gene (lncRNA), and16.61%pseudogene.The results indicate complex dysregulation of the cellular transcriptome in PCa.
2.Somatic DNA copy number aberrations in prostate cancer cell genome.
We analyzed the somatic copy number alterations (SCNAs) across258tumor-normal paried samples. Data from high-resolution single nucleotide polymorphisms (SNP) microarray on258PCa specimens and the matching normal specimens were retrieved from TCGA.We found2448genes with high-frequency gain and loss, including961PCGenes,808lncRNAs, and679pseudogenes.
3. Epigenetic analysis of PCa.
To analyze DNA methylation alterations in PCa, we obtained DNA methylation microarray profiles (Infinium Human Methylation450BeadChip) on252human tumor and49normal control specimens from TCGA. We finally identified2617cancer-associated epigenetically silenced genes. The results indicatePCa is characterized by genome-wide methylation imbalance. Next, we performed analysis on miRNA (miRNA-seq) expression profiles of in259human tumor and50normal control specimens from TCGA. We identified36.90%miRNAs that were up-regulated or down-regulated in the tumor specimen compared to normal specimen.
二、Analysis of expression and functional modules of AGR2based on bioinformatic and experimental data.
1. Up-regulation of AGR2in PCa samples.
AGR2is expressed in relatively high levels for prostate cancer patients based on TCGA data. DNA copy number and global methylation analysis showed there were no copy number variations or methylation dysregulation of AGR2in PCa samples. So enhanced mRNA stability and constitutive activation of transcription factor may be involved in upregulation of AGR2.
2. AGR2was overexpressed in Chinese Han PCa tissues.
An elevated expression for AGR2has been observed in prostate tumor tissues of Europe and USA men, however, little is known about the expression level in human tissues among the Chinese Han population. Immunohistochemical analysis of AGR2expression was performed in BPH tissues from41patients, PIN tissues from13patients, and PCa specimens from32patients. The data showed AGR2was overexpressed in Chinese Han PCa tissues as well. We then evaluate the differential expression of AGR2at the protein levels in a panel of various cell lines, and the resluts showedthe expression of AGR2was significantly up-regulated in PCa cells than normal prostate epithelial cells.
3. Analysis of functional modules of AGR2based on TCGA data
we performed Guilt-by-Association (GBA) analysis for AGR2on RNASeq profiles in TCGA to characterize the biological functions and the molecular pathways influenced by AGR2-associated genes. The results showed AGR2correlates with cell cycle, DNA damage and repaired, and cell adhesion.
三、AGR2i-induced DNA damage is involved in regulation of cellular senescence in PCa.
1. Knockdown of AGR2inhibited proliferation in PCa cells.
Knockdown of AGR2by AGR2-targeting siRNA caused a decrease in cell viability in PC3cells, whereas forced expression of AGR2led to an increased cell proliferation of LNCaP and DU145cells.We further assessed the effect of AGR2in cell proliferation process by analysis of incorporation of BrdU into DNA after silencing the endogenous AGR2, and BrdU signaling in AGR2i-treated PCa cells was significantly decreased. Western blotting results showed inactivation of AKT and ERK was accordingly observed in AGR2i-treated cells.
2.Knockdown of AGR2induced cell cycle arrest in PCa cells.
AGR2was overexpressed in Chinese Han PCa tissues and had a positive correlation with Cyclin D1and p-Rb, but not with p16.AGR2-depletion resulted in accumulation of cells at the G0/G1phase.
3. Knockdown of AGR2induced cellular senescence in PCa cells.
AGR2siRNA treatment resulted in cellular senescence in all three PCa cell lines as indicated by an increase of flat, enlarged and senescence-associated β-galactosidase (SA-β-Gal) positive cells. Senescent response to AGR2-silencing was also evidenced by elevated fluorescent punctuate formation of tri-methyl-histone H3in AGR2-depleted cells.
4. AGR2depletion-induced cellular senescence depends on CDKIs.
Further studies indicated that LNCaP underwent a p21-dependent cellular senescence in response to AGR2depletion that requires inactivation of ERK signaling, while PC3was also p21-dependent, but involved in suppression of PI3K/Akt. Unlike LNCaP and PC3, senescent response of DU145was found to be mainly p27-dependent that may require upregulation of PTEN and inhibition of PI3K/Akt signaling.
5. AGR2depletion-induced cellular senescence was accompanied with DNA damage.
Senescence is considered as a DNA damage responseriggered by DNA hyper-replication or other cellular stress. Cells were stained with γH2AX-FITC antibodies and subjected to flow cytometryto quantitatively determine the expression levels of the DNA damage marker γH2AX. Senescent response to AGR2-silencing was evidenced by elevated γH2AX, which means AGR2i-induced DNA damage may be involved in regulation of cellular senescence.
Part Ⅱ DNA damage response indeuced by bisbibenzyls is involved in regulation of cellular senescence and apoptosis in prostate cancer
We found AGR2depletion induced DNA damage and resulted in cellular senescence in PCa cells, and cellular senescence was shown to be one of the key determinants of tumor response to therapy in vitro and in vivo.The concept of pro-senescence therapy has emerged over the past few years as a novel therapeutic approach to treat cancers. Damage to DNA, the prime target of anticancer therapy, triggers programmed cellular responses. In addition to apoptosis, therapy-mediated cellular senescence has been identified as another drug-responsive program that impacts the outcome of cancer therapy.
One of the most efficient chemical reagents used in cancer chemotherapy are DNA damage inducers, which can cause a variety of DNA lesions via multiple mechanisms and resulted in cellular senescence and apoptosis. For example, camptothecin and etoposide can trigger single-strand breaks (SSBs) or double-strand DNA breaks (DSBs) by trapping topoisomerase-DNA covalent complexes, subsequently leading to the cell death; cisplatin and its derivatives forms DNA cross links as monoadduct, interstrand crosslink, intrastrand crosslink or DNA protein crosslink, thus induced DNA damage; cyclophosphamide alkylates the N-7position of guanine and results in frameshift mutation. Cells exposed to chemicals are prone to acquire multiple sites of bulky DNA. Moreover, DNA damaging agents can damage other biomolecules such as proteins, carbohydrates, lipids, and RNA. The accumulation of damage, to be specific, double-strand breaks or adducts stalling the replication forks, are among known stimulation signals for a global response to DNA damage. The global response to damage is an act directed toward the cells' own preservation and triggers multiple pathways of macromolecular repair, lesion bypass, tolerance, senescence, or apoptosis. The common features of global response are induction of multiple genes, cell cycle arrest, and inhibition of cell division.
Depending on the type of DNA lesions, specific cell cycle checkpoints and cellular cascades are activated by DNA-damaging agents. Checkpoint activation pauses the cell cycle and gives the cell time to repair the damage before continuing to divide. As widely accepted, ataxia telangiectasia mutated (ATM) and ataxia telangiectasia and Rad3related (ATR) signaling pathways play important roles in response to DNA damage response. ATM responds mainly to DSBs, and initiates phosphorylation of downstream targets such as Chk2, BRCA1, and NBS1proteins at the site of DNA damage. These factors act together to induce G1, S, and G2cell cycle arrests, DNA repair, and/or activation of cell death pathways. While ATR is activated in response to replication stress, it triggers the activation of Chkl, which in turn leads to the phosphorylation of Cdc25and prevents the activation of CDK1/Cyclin B and mitotic entry. Upon DSBs, the process of DSBs end joining involves numerous proteins and enzymes through nonhomologous end joining (NHEJ) and homologous recombination (HR) repair mechanisms. For example, the Ku70/86heterodimer is critical in NHEJ, since it binds to the broken DNA ends and recruits repair-related proteins including DNA-dependent protein kinase, XRCC4, and DNA Ligase Ⅳ. The rate of DNA repair is dependent on many factors, including the cell type, the age of the cell, and the extracellular environment. A cell that has accumulated a large amount of DNA damage, or one that no longer effectively repairs damage incurred to its DNA, can enter one of three possible states:an irreversible state of dormancy, known as senescence, or cell suicide, also known as apoptosis.
一、Low concentrations of RD triggered p21dependent cellular senescence.
1. Low concentrations of RD induced cellular senescence.
0.5μmole/L RD treatment caused growth arrest at G0/G1phase with features of cellular senescence phenotype such as enlarged and flattened morphology, increased senescence-associated-beta-galactosidase staining cells, and decreased cell proliferation in PCa cells. Induction of cellular senescence by RD occurred through activation of DNA damage response including increases in the γH2AX,inactivation of Chk1/2, and suppression of repair-related Ku70/86and phosphor-BRCA1in PCa cells.
2. RD-triggered cellular senescence is p21denpendent.
It has been demonstrated that the p21-Rb signaling pathway, activated by DNA damage response, plays an important role in the activation of G0/G1checkpoint response and cellular senescence.We then detected the activation of p21in PCa cells, and found the expression of the p21protein was markedly enhanced. We next explored a link between the enhanced p21 expression and senescence induction in response to RD by genetic knockdown of p21. The results revealed that deletion of p21by targeting siRNA impaired RD-induced senescent cells in all three PCa cells, indicating that RD-induced cellular senescence was, at least in part, in a p21-dependent manner.
3. RD triggers cellular senescence in PC3xenograft.
To determine whether RD could induce tumor cell senescence in vivo, as observed in cultured cells, human PC3xenografts were developed in male nude mice. Administration of RD had no effect on body weight but significantly reduced tumor growth of tumor-bearing mice.To examine whether decreases in tumor mass and size by RD were associated with DNA damage-mediated cellular senescence, tissue samples were prepared for immunohistochemical analysis. Consistent with results in culture cells, senescent cells were pronounced in RD-treated tumor tissues compared to the placebo treatment, as evidenced by increased SA-P-Gal staining, associated with substantial accumulation of ting nand positive stained tri-Me-K9-H3which serve as a senescence-associated heterochromatic foci. Meanwhile, RD-mediated alterations in the expressions of p21were similar to the observations shown in culture cells. These data demonstrated that RD triggered DNA damage and cellular senescence that contributed to its observed antitumor effect.
二、RD triggered DNA damage and inhibited DNA repair in PCa cells.
1. RD caused remarkable DNA damage in PC3cells.
After24h treatment with10μmol/L RD, cell cycle was significantly arrested in G2/M phase in PC3cells. In addition to interference with cell cycle progression, RD was able to noticeably induce apoptosis as well. As evidenced by induction of yH2AX foci, micronuclei, and DNA fragmentation in Comet assay, we found RD caused remarkable DNA damage in PC3cells. Time kinetic and dose-dependent studies showed that ATM/Chk2and ATR/Chkl signaling pathways were sequentially activated in response to RD. Blockage of ATM/ATR signaling led to the attenuation of RD-inducedyH2AX, and to the partial recovery of cell proliferation.
2. RD inhibited DNA repair in PC-3cells
Western blotting data showed RD exposure resulted in the inactivation of BRCA1which is a critical molecule in the initial recruitment of other repair proteins/enzymes at the DNA breaks. To determine the effects of RD on DSBs repair, the ability of DNA repair was evaluated with HR and NHEJ reporters. Positive signal significantly declined in either NHEJ or HR repair systems in cells treated with RD, indicating that DSBs repair was impaired in response to RD. Based on the observations above, we further clarified the role of Ku70/Ku86in response to RD-induced DNA damage. DNA end-binding activity of Ku70/Ku86displayed that, compared with the untreated cells, the binding activities of both Ku70/Ku86in treated cells were significantly decreased after12h treatment. Microarray analysis revealed that genes related to damaged-DNA binding, DNA repair, cell cycle, and apoptosis were significantly changed in RD-treated cells.
3. RD triggers apoptosis associated with the induction of DNA damage in PC3xenograft.
Administration of RD to xenograft mice reduced tumor growth, and coordinately caused alterations in the expression of genes involved in DNA damage and repair, along with cell apoptosis.Changes of molecular markers associated with DNA damage in treated mice displayed that RD caused DNA-damage response via ATM/ATR-mediated signaling as evidenced by substantial accumulation of yH2AX, abrogation of phosphor-BRCAl, and Ku70/Ku86abundance. The data demonstrated that RD triggered DNA damage and impaired repair proteins, leading to apoptotic cell death, which contributed to its antitumor effect.
Part III The molecular mechanism of Bisbibenzyls-induced apoptosis in androgen dependent prostate cancer cells
In the last half of the century, advances in tumor cell biology and molecular biology of cancer have been responsible for achievements in cancer therapy. Currently, docetaxel is as an active agent to moderately improve quality of life and survival conditions in patients with metastatic HRPC. However, only about50%of patients treated with docetaxel have a prostate-specific antigen response. Hence, many efforts have been made to investigate novel mechanisms of action of bioactive chemicals with less toxicity and high efficacy to treat HRPC. Macrocyclic bisbibenzyls are a series of phenolic natural products isolated from liverwort, which exert a variety of biological activities, including antifung, antioxidation and cytotoxicity. Our previous research demonstrates its inhibitory effect on PCa cells.
Currently, PI3K pathway is considered a key regulator of progression to androgen-independent prostate cancer, while in androgen-dependent prostate cancer androgen receptor (AR) signaling pathway plays key role in maintaining development, proliferation and differentiation in prostate gland. AR, a nuclear receptor, dissociated from heat shock protein when it bind its ligand, which in turn promotes AR nuclear location and dimerization. Activated AR binds to AR response elements (AREs) and regulates gene transcription, such as PSA. Previous report displayed that AR promoted development from hormone-dependent prostate cancer to horomone-independent prostate cancer. Abnormal androgen signaling due to aberrant expression mutations, or dysregulation of the AR gene has been linked to prostate tumorigenesis, and progression of prostate cancer into advanced, castration-resistant disease. Treatment of androgen-dependent LNCaP cells with macrocyclic bisbibenzyl compound Riccardin C (RC), Marchantin M (MM) or Riccardin D (RD) caused significantly cell cycle arrest and apoptosis. AR is a key factor for cell proliferation in LNCaP cells, so we investigated whether bisbibenzyl compound involved in inhibiting AR function and triggering apoptosis in LNCaP cell.
一、Macrocyclic bisbibenzyl compounds induced apoptosis by inhibiting AR expression and function in LNCaP cells.
1. Exposure of androgen-denpendent cell line LNCaP to RC, MM, or RD caused markedly suppression of cell proliferation.
Treatment of LNCaP cells with RC, MM or RD caused cell cycle arrest in G0/G1phase associated with moderate increases of p53, and p21protein levels, and significantly triggered apoptosis with induction of a higher ratio of Bax/Bcl-2, and elevated cleavage of PARP.
2. Induction of apoptosis was accompanied with the decreased expression and transcriptional activity of the AR.
As demonstrated by western-blotting analysis, AR expression was significantly repressed in LNCaP cells exposed to RC, MM and RD. Luciferase activity assays showed RC, MM and RD treatment markedly suppressed androgen-mediated expression of the AR promoter activity. We further examined the impact of bisbibenzyls on AR transcriptional activity by monitoring the activity of the promoter of prostate specificantigen (PSA), a well-known AR target gene, and found PSA promoter was dramatically repressed by RC, MM and RD in LNCaP cells. We then examined SRC-1/AR interactions using co-immunoprecipitation (co-IP), and declined SRC-1proteins were noticeably observed in cells treated with RC, MM and RD.Thus, inhibitory effect of bisbibenzylson cell proliferation was associated with the downregulation of AR expression,leading to the abolishment of AR.
3. RC, MM and RD targeting proteasome activity promotes autophagy inLNCaP cells.
Proteasome activity assays with purified proteasome in the presence of bisbibenzyls were performed, and the results revealed that RC, MM and RDsignificantly inhibited the proteasome. Werstern blotting and co-IP results demonstrated that the levels of polyubiquitinated-AR were noticeably increased in LNCaP cells treated with RC, MM or RD. We hypothesized that bisbibenzyl-mediated proteasome inhibition simultaneously activates autophagy in LNCaP cells, which in turn is involved in degrading the polyubiquitinated-AR. In our study, induction of LC3B expression and conversion was found in LNCaP cells treated with the compounds which demonstrated the ability of autophagy induction by these agents. We then performed immunofluorescence co-location analysis on AR and LC3or Ub, and the results showed a co-localization of AR and LC3or Ub, suggesting that activation of autophagy by RC, MM and RD might be involved in the process of ubiquitinated ARdegradation.
Part IV Conclusions and Innovation
一、Conclusions
1. A map of prostate cancer genome variation revealed characteristic differences between normal prostate and prostate cancer.
2. AGR2overexpressed in PCa, and knockdown of AGR2caused cellular senescence in PCa cells.
4. Riccardin D induced DNA damage-mediated cellular senescence and apoptosis in prostate cancer.
5. Macrocyclic bisbibenzyl compounds induced apoptosis by inhibiting AR expression and function in androgen-denpendent cell line LNCaP.
二、Innovation
1. With the multi-omic profiles and well-annotated clinical and pathological information on large-scaled clinical samples, we presented the genomic landscape of PCa and provided a powerful resource to improve our ability to diagnose, treat, and prevent cancer through a better understanding of the genetic basis of this disease.
2. We firstly reported AGR2was overexpressed in Chinese Han PCa tissues, and knockdown of AGR2caused cellular senescence in PCa cells.
3. We firstly reported macrocyclic bisbibenzyl compounds prevented AR expression and function, and promoted apoptosis in LNCaP cells. Since AR in LNCaP cells was mutant, the characteristics of bisbibenzyl will be beneficial for the treatment of prostate cancer expression constitutive activation of the AR.
4. We firstly reported RD triggered DNA damage and inhibited DNA repair, thus induced cellular senescence or apoptosis in prostate cancer. Significantly, DNA repair contributes to the drug-resistant phenotype, so that RD may reverse the drug-resistant phenotype by inhibiting repair of chemotherapy-induced genotoxic damage. This finding provides the foundation for the use of RD as a chemotheraputic candidate.
PCa作为激素依赖性肿瘤,去势治疗是首选标准方案,约80%的前列腺癌对去势治疗敏感。然而绝大多数PCa患者经去势治疗1-2年后,肿瘤复发,成为激素难治性前列腺癌(Hormone Refractory Prostate Cancer, HRPC),抵抗内分泌治疗,抗凋亡能力强,伴随肿瘤的侵袭转移,死亡率高,预后差。由于HRPC的发展和演变是复杂的多因素、多信号通路参与的过程,目前的研究主要侧重于两方面,一是HRPC发生发展的机制,二是新靶点的鉴定及其干预药物。我们利用生物信息学技术,通过分析癌症和肿瘤基因图谱(Cancer Genome Atlas, TCGA)计划所提供的大规模基因组测序数据,绘制了人前列腺癌的基因组变异图谱,并进行系统分析,确定与HRPC的分期、侵袭转移、药物耐受等恶性表型密切相关的基因,筛选出AGR2为候选靶基因之一。由于TCGA的数据基础主要来自欧美人群,我们又利用中国汉族人群PCa患者的临床样本进行分析,发现AGR2与中国汉族人群PCa患者的恶性表型密切相关。进一步研究发现,降低AGR2在PCa细胞中的表达,可通过DNA损伤应答机制诱导细胞衰老。同时发现具有拓扑异构酶抑制活性的天然联苄化合物,可通过DNA损伤应答信号诱导PCa细胞衰老,而在DNA损伤程度较强时诱导激素非依赖PCa细胞凋亡。另外还发现,联苄化合物可通过抑制雄激素受体的表达和功能诱导激素依赖性PCa细胞凋亡。
第一部分基于前列腺癌基因组图谱的AGR2功能分析与AGR2缺乏介导的DNA损伤诱导前列腺癌细胞衰老的机制作用
目前已知的癌症有200多种,但是所有肿瘤在特殊类别(分型)或发展的不同分期方面都发现有基因组的特异变化,而正是基因组的改变导致了细胞分化、发育和生长通路的异常,从而引发细胞增殖、生长的失控。本论文通过分析癌症和肿瘤基因图谱(Cancer Genome Atlas,TCGA)计划所提供的大规模基因组测序数据,绘制了人类前列腺癌的基因组变异图谱,并进行系统分析,旨在找到所有癌基因和抑癌基因的微小变异,分析其在前列腺癌发生、发展的变化趋势及可能的作用,确定能够驱动PCa发生的潜在drivers或治疗靶点,以期发现前列腺癌发生发展和治疗的新策略。
前梯度同源蛋白2(Anterior gradient protein2homolog, AGR2)是本课题组通过前列腺癌基因组图谱分析得到的候选靶基因之一。现已报道多种肿瘤的发生与发展伴随着AGR2表达的上调,包括前列腺癌、乳腺癌、肺癌和胰腺癌等等。在正常的前列腺组织中,AGR2受雄激素诱导表达,但是对前列腺癌样品的显微切割分析表明AGR2的mRNA和蛋白水平都较临近的良性组织有显著的升高。最近有报道指出,在前列腺癌患者的尿液中检测到高水平的AGR2,因此AGR2有望作为前列腺癌的尿检标志物。到目前为止,尚未报道过汉族人群前列腺癌组织中AGR2蛋白的表达情况及作用机制。本课题组对汉族人群前列腺组织AGR2蛋白的表达做了检测,发现在汉族人群前列腺癌组织中AGR2的表达水平显著高于良性组织,同时在前列腺癌细胞系中也观察到AGR2的表达明显高于前列腺正常上皮细胞,并首次发现和提出抑制前列腺癌细胞中AGR2的表达可导致显著性细胞衰老。通过详细剖析AGR2基因在前列腺癌细胞中的生物学功能,表明AGR2在前列腺癌检测和治疗中具有潜在价值。
一、前列腺癌基因组图谱的绘制
1.前列腺癌转录组严重失调。通过对256例前列腺癌组织以及45例前列腺癌旁正常组织的转录组测序(Whole Transcriptome Shotgun Sequencing, RNASeq)数据分析,癌组织中24%以上的基因较正常组织有显著性上调或下调,其中蛋白编码基因(Protein-coding genes, PCGene)失调率为28.53%,长链非编码RNA(long non-coding RNA,lncRNA)失调率为27.64%,假基因(Pseudogene)失调率为16.61%。
2.前列腺癌体细胞基因组中存在DNA序列拷贝数的畸变。通过对258例前列腺癌样本以及相匹配的正常对照组织的高分辨率单核苷酸多态性基因分型芯片(Affymetrix Genome-Wide Human SNP Array6.0,SNP6.0)分析,共找到了2448个拷贝数异常的基因,包括961个PCGene,808个lncRNA和679个Psuedogeneo
3.前列腺癌表观遗传学分析。通过对252例前列腺癌组织以及49例前列腺癌旁正常组织的全基因组甲基化芯片(Illumina Infinium HumanMethylation450BeadChip)分析,得到了2617个前列腺癌特异性过甲基化基因,提示前列腺癌中存在全基因组甲基化失衡。对259例前列腺癌组织以及50例前列腺癌旁正常组织的：miRNA深度测序(miRNASeq)数据分析显示,前列腺癌组织中36.90%的miRNA较正常组织有显著性上调或下调。
二、基于图谱信息和实验数据对AGR2的表达和功能的分析
1.AGR2基因在PCa组织中表达显著上调。通过对前列腺癌INASeq数据分析,发现AGR2基因在前列腺组织中有较高的基础丰度,而且在PCa组织中的表达丰度上调非常显著。而DNA序列拷贝数数据分析结果表明,AGR2的拷贝数在正常组织和癌组织中并无显著差异。另外,全基因组甲基化芯片分析结果显示,AGR2在肿瘤组织中并无异常甲基化修饰。这些结果表明,AGR2在PCa中的高表达并非拷贝数增加或甲基化异常,而mRNA稳定性增加、或在转录水平表达增强等机制可能参与其调控,尚需进行研究。
2.AGR2在中国汉族人群PCa患者的组织中表达显著上调。由于TCGA的数据和文献报道主要来自欧美国家人群,而PCa的发生与种族有关。到目前为止,中国汉族人群前列腺癌患者组织中AGR2蛋白的表达情况及功能尚未报道。本课题通过免疫组化技术分析汉族人群良性前列腺增生、前列腺癌组织中AGR2蛋白的表达情况,结果显示,汉族人群前列腺癌组织中AGR2的表达水平显著高于良性组织,与欧美人群的表达趋势一致。Western blotting结果显示,AGR2在前列腺癌细胞系中的表达明显高于前列腺非恶性上皮细胞。因此,上述结果显示,AGR2基因在前列腺癌组织和细胞中的表达调控、生物学功能都有待进一步研究,以阐明其在前列腺癌发生发展和治疗中的潜在价值。
3.AGR2在前列腺癌中的功能。基于RNASeq数据组对AGR2的连坐效应相关分析(Guilt-by-Association,GBA)提示,AGR2在前列腺癌中的功能可能与细胞周期、DNA损伤修复、细胞粘附等相关。文献已有报道,AGR2与细胞的增殖、侵袭转移等有关,但机制尚需进一步研究。
三、DNA损伤应答参与AGR2介导的前列腺癌细胞衰老的机制研究
1.下调AGR2抑制PCa细胞增殖。上述结果表明,AGR2在中国汉族人群PCa组织、细胞系中高表达,我们首先在AGR2表达相对较高的PC3细胞中通过RNAi下调AGR2的表达,观察细胞表型的变化。MTT结果显示,下调AGR2后,显著抑制PC3细胞的增殖。相反,在AGR2表达相对较低的LNCaP和DU145细胞中转染AGR2的表达质粒,细胞的存活数显著增加。BrdU掺入实验结果显示,三种PCa细胞中下调AGR2的表达后,BrdU的掺入显著降低,表明AGR2促进细胞增殖。Western blotting结果显示,下调AGR2后,抑制PCa细胞增殖、存活信号通路,如p-Akt表达水平下降,但Akt总蛋白的表达水平并无变化；p-ERK的水平略有下调,对总ERK蛋白的表达水平无影响。
2.下调AGR2阻滞PCa细胞周期进程。免疫组化结果显示,PCa组织中AGR2的高表达伴随着周期蛋白Cyclin D1的表达上调,而周期抑制因子p16、pRb在良性前列腺增生、前列腺内皮肉瘤中表达升高,但在PCa组织中表达显著下调。IINAi结果显示,下调AGR2的表达,PCa细胞显著阻滞于G1期。
3.抑制AGR2的表达诱导前列腺癌细胞衰老。利用siRNA抑制AGR2的表达后,PCa细胞扁平胀大呈现明显的细胞衰老形态。衰老相关β-半乳糖苷酶(senescence associated P-galactosidase,SA-β-Gal)染色和衰老相关异染色质聚集灶(Senescence-associated heterochromatin foci,SHAF)分析进一步确认抑制AGR2表达可诱导前列腺癌细胞衰老。
4.干扰AGR2的表达上调细胞周期抑制因子从而引起细胞衰老。在前列腺癌细胞系LNCaP(表达野生型p53)和PC3(p53无)中抑制AGR2表达后,p21以p53非依赖的方式表达上调,Cyclin D1、pRb的表达显著下降,p16的表达有所增加。RNAi实验结果表明,下调AGR2后诱导的LNCaP和PC3细胞衰老过程依赖于p21的激活。而在DU145细胞中(表达野生型PTEN,无功能性p53),下调AGR2的表达,使PTEN的表达有所增加,显著诱导p27的表达,而p16的表达有所增加,p21的变化不显著。进一步分析证实下调AGR2诱导的DU145细胞衰老则部分依赖于p27信号通路。
5.抑制AGR2的表达诱导细胞衰老伴随DNA损伤。由于DNA损伤应答信号是诱导细胞衰老的主要机制之一,我们利用荧光抗体标记、流式分析DNA损伤的标志蛋白yH2AX的变化,结果显示,下调AGR2表达后,LNCaP和PC3细胞中yH2AX的表达显著增加,DU145细胞中yH2AX表达上调,但较LNCaP和PC3细胞弱,表明,DNA损伤参与AGR2下调诱导的细胞衰老过程,但机制尚需进一步探讨。
第二部分DNA损伤应答抑制DNA损伤修复引起细胞衰老或凋亡的机制研究
由于下调癌基因AGR2的表达,可诱导PCa细胞衰老,使我们进一步分析诱导细胞衰老作为PCa治疗的可能性。由于衰老细胞缺乏增殖能力,这无疑有利于肿瘤的治疗,因此诱导肿瘤细胞衰老(Prosenescence therapy)成为近年来的研究热点。研究发现,化疗(如DNA损伤药物顺铂、多柔比星等)后,肿瘤组织中除了凋亡细胞外,也产生衰老细胞,称为TIS (Therapy-Induced Senescence)。
DNA损伤药物是目前研究较多的促进细胞衰老的诱导剂。药物诱导的DNA损伤类型有多种,例如喜树碱和依托泊苷可以通过捕获拓扑异构酶-DNA复合物触发DNA单链或双链的断裂；顺铂可与DNA结合,产生链内交联与链间交联,从而破坏DNA的功能；环磷酰胺可使DNA分子中的鸟嘌呤N7烷化后除去,留下缺失碱基后的空隙,造成移码突变。并且一种DNA损伤剂往往可以同时引起几种类型的损伤,其损伤效应的大小和类型与剂量及细胞所处的周期状态有关。
对于不同的DNA损伤病变,特异的DNA损伤检控点通路将被激活。DNA损伤检控点通路是由一系列检控蛋白构成的复杂的信号转导网络,在细胞周期转换、DNA复制和染色体分离中发挥重要的作用,并对DNA损伤做出及时应答启动修复,以防止基因突变和遗传不稳定的发生。研究表明,毛细血管扩张性共济失调症突变蛋白(ataxia-telangiectasia mutated, ATM)、毛细血管扩张性共济失调症突变Rad3相关蛋白(ATM and Rad3-related,ATR)在DNA损伤应答系统中起到了至关重要的作用。ATM主要应对双链断裂,当DNA双链受损时,ATM发生自磷酸化并激活一系列下游分子,包括Chk2, BRCA1和NBS1等,在这些因子的共同作用下,细胞的应激系统被启动,产生周期阻滞,并进行DNA修复,细胞衰老和凋亡也可能随之发生。当细胞受到严重的DNA复制压力时ATR将被激活,并磷酸化Chkl,激活下游Cdc25分子,抑制CDK1/Cyclin B复合物活性使细胞周期阻滞,进而启动DNA修复程序。DNA双链断裂主要有两种修复机制,同源重组(homologous recombination,HR)和DNA末端链接(nonhomologous end joining,NHEJ)。HR修复是利用细胞内的染色体两两对应的特性,若其中一条染色体上的DNA发生双股断裂,则另一条染色体上对应的DNA序列即可当作修复的模版来回复断裂前的序列；NHEJ修复不需要任何模版,修复蛋白Ku70/86可以直接将双股裂断的末端彼此拉近,再借由DNA黏合酶的帮助下,将断裂的两股重新接合。DNA损伤检控点信号转导通路功能和DNA修复机制异常,DNA损伤不能及时得到修复,细胞将会走向衰老或凋亡。本课题组研究发现,双联苄化合物RD,能够诱导前列腺癌细胞DNA损伤,抑制DNA损伤修复,最终诱导前列腺癌细胞走向衰老或凋亡。
一、低剂量片叶苔素D诱导前列腺癌细胞产生p21依赖的细胞衰老
1.低剂量RD诱导前列腺癌细胞衰老。0.5μmol/L的RD处理细胞10天后,前列腺癌细胞PC3、DU145和LNCaP均出现细胞克隆能力显著下降、周期阻滞、细胞形态扁平胀大等细胞衰老特征。衰老相关β-半乳糖苷酶染色和衰老相关分泌表型(enescence-associated secretory phenotype,SASP)分析证实,RD诱导前列腺癌细胞产生明显的细胞衰老。DNA损伤标志蛋白yH2AX的免疫荧光结果显示,低剂量的P.D诱导前列腺癌细胞产生了明显的DNA损伤,Western-blotting结果显示,DNA损伤检控点蛋白Chkl/2和损伤修复相关蛋白Ku70/86与p-BRCAl表达受到抑制。以上结果提示,低剂量RD可能通过诱导引起前列腺癌细胞DNA损伤并抑制损伤修复,从而诱导细胞衰老。
2.低剂量RD诱导的细胞衰老依赖于p21的激活。Western-blotting和实时定量PCR结果显示,RD处理后,前列腺癌细胞p21的表达显著增高。利用siRNA特异性的降低p21的表达,可显著逆转RD诱导的细胞衰老。
3.RD诱导荷瘤动物体内癌细胞衰老。裸鼠荷瘤实验表明,RD能显著抑制荷瘤动物体内癌细胞生长。衰老相关β-半乳糖苷酶染色和衰老相关异染色质聚集灶分析表明,RD处理后,裸鼠体内癌细胞出现明显的细胞衰老。DNA损伤标志蛋白γH2AX的免疫组化结果显示,RD诱导裸鼠体内癌细胞出现明显的DNA损伤。免疫组化结果显示p21在RD处理组的裸鼠癌细胞中的表达显著上调,提示RD诱导裸鼠体内癌细胞衰老的机制可能与p21的激活有关。
二、片叶苔素D诱导前列腺癌细胞DNA损伤并抑制损伤修复
1.片叶苔素D诱导前列腺癌细胞DNA损伤,激活损伤应答程序。细胞周期分析结果显示,终浓度10μmol/L的RD处理细胞24h后,前列腺癌细胞细胞周期被明显阻滞在G2/M期,并伴随大量的细胞凋亡。yH2AX焦点分析、微核试验和彗星实验结果均证实,10μmol/L的RD诱导前列腺癌细胞PC3产生明显的DNA损伤。时相分析显示,DNA损伤检控点通路ATM/Chk2和ATR/Chkl在处理1h后显著激活。ATM/ATR抑制剂处理细胞后,能部分逆转片RD导致的细胞增殖抑制。
2.片叶苔素D显著抑制DNA损伤修复。Western-blotting结果显示10μmol/L的RD显著抑制DNA损伤修复相关蛋白BRCA1的表达；DNA修复活性检测表明,RD显著抑制了HR和NHEJ修复系统功能,并抑制了DNA修复酶Ku70/86的表达和活性。基因芯片结果分析显示,受RD影响的基因主要集中在细胞增殖,细胞周期,DNA损伤和修复,以及凋亡相关通路。
3.片叶苔素D诱导荷瘤动物癌细胞DNA损伤。裸鼠荷瘤实验表明,RD能显著抑制荷瘤动物体内癌细胞生长。Western-blotting和免疫荧光结果显示,RD能够改裸鼠体内癌细胞DNA损伤修复以及细胞凋亡相关通路基因的表达。其中DNA损伤标志蛋白yH2AX的显著上调表明,RD处理的裸鼠癌细胞存在明显的DNA损伤。
第三部分双联苄化合物抑制雄激素依赖性前列腺癌细胞增殖机制研究
随着肿瘤细胞生物学和肿瘤分子生物学研究的发展,人类对癌症的认识越来越深入,在肿瘤治疗方面取得了很多振奋人心的成果。例如：小分子化合物多西紫杉醇对去势治疗失败的转移性前列腺癌患者显示出明显生存优势,极大的改善了晚期前列腺癌患者的生存期。然而过半的前列腺癌患者存在药物不敏感及耐药现象,因而亟待研发新的抗肿瘤药物。双联苄(Bisbibenzyls)是从苔藓植物分离提纯得到的一类大环多酚类化合物,这类化合物结构新颖、多变,联苄之间具有连接方式和取代基团多样化的特点。前期研究发现这种化合物具有抗肿瘤、抗炎、抗真菌等多种生物活性,具有良好的开发应用潜力和前景。本课题组基于双联苄类化合物片叶苔素C(Riccardin C)、地钱素M(Marchantin M,MM)和片叶苔素D(Riccardin D)对雄激素依赖性细胞系LNCaP的增殖抑制作用,研究了其具体机制。
近年来研究显示,P13K信号途径对雄激素非依赖性前列腺癌的发生起重要作用,而AR信号通路则是激素依赖性前列腺癌增殖的关键途径。雄激素受体(Androgenreceptor,AR)是一种核受体型转录因子,当雄激素与AR结合后,会引起AR构象的改变,使AR与热休克蛋白90(HSP-90)解离,随即活化而转移至细胞核内,并形成同源二聚体与雄激素受体应答元件(AREs)结合,诱导下游靶基因的转录表达,促进细胞增殖。因此,有效抑制雄激素依赖的前列腺癌中AR的表达和功能一直是倍受关注的热点话题。本课题研究结果显示,双联苄化合物片叶苔素C(Riccardin C)、MM和RD能显著抑制雄激素依赖的前列腺癌细胞LNCaP的增殖能力。由于AR是LNCaP细胞增殖存活的关键调控因子,我们研究了双联苄化合物对AR表达和功能的影响以及与LNCaP增殖抑制之间的关系。
一、双联苄化合物抑制AR的表达和功能促进LNCaP细胞凋亡
1.RC、MM和RD抑制雄激素依赖的前列腺癌细胞LNCaP恶性增殖。RC、MM和RD将LNCaP细胞周期阻滞在G0/G1期,并伴随着p53和p21蛋白水平的增高；Western-blotting结果显示,双联苄化合物处理后Bax/Bc12蛋白比例和PARP剪切形式明显增加,表明LNCaP细胞产生明显的凋亡。
2. LNCaP细胞凋亡伴随着AR表达和活性的降低。荧光素酶报告基因分析和Western-blotting结果显示,RC、MM和RD明显抑制LNCaP细胞AR的转录活性和蛋白表达。当双联苄化合物处理后,LNCaP细胞中PSA启动子活性降低,免疫共沉淀(co-IP)结果显示AR与SRC-1结合减弱,提示AR的功能受到明显抑制。
3.双联苄化合物抑制蛋白酶体活性诱导LNCaP细胞自噬。RC、MM或RD处理细胞24h后,蛋白酶体活性显著下降,Western-blotting和co-IP结果显示,泛素化AR在细胞内蓄积。LC3蛋白表达及免疫荧光分析证实双联苄化合物引起LNCaP细胞自噬,AR、 LC3及Ub的公定位分析提示,泛素化AR的降解与细胞自噬有关。
第四部分结论及创新点
一、结论
1.前列腺癌组织基因组较正常组织出现了明显的变异。
2.AGR2在前列腺组织中高表达,抑制AGR2诱导前列腺癌细胞衰老。
4.片叶苔素D诱导前列腺癌细胞DNA损伤并抑制损伤修复,引起前列腺癌细胞衰老或凋亡。
5.双联苄化合物能够抑制激素依赖的前列腺癌细胞AR的表达和功能,从而抑制其恶性增殖。
二、创新点
1.利用大样本、多平台、高通量的基因组学手段,细致绘制了前列腺癌基因图谱。为研究前列腺癌细胞发生、发展机制,探索新的诊断和治疗方法,提供了坚实的理论基础。
2.首次报道AGR2在汉族人群前列腺癌患者中的表达分布,首次发现和提出在前列腺癌细胞中抑制AGR2的表达可诱导癌细胞衰老。
2.首次报道双联苄化合物抑制AR的表达和功能,进而促进细胞凋亡。由于LNCaP细胞表达的是突变型AR,双联苄化合物的这种抑制作用将有益于AR持续活化的前列腺癌的治疗。
3.首次报道片叶苔素D可诱导前列腺癌细胞DNA损伤并抑制损伤修复,从而导致癌细胞衰老或凋亡。由于DNA损伤修复机制是大多数DNA损伤类化疗药物敏感性的关键因素之一,片叶苔素D不仅能诱导前列腺癌细胞DNA损伤,并能进一步抑制损伤的修复,这些特点将使片叶苔素D成为很有潜力和竞争力的抗肿瘤候选药物。
Rates of detection of prostate cancer (PCa) vary widely across the world, and PCa is the most common cancer in men in the Western countries. While improved early detection significantly decreased mortality, PCa still remains the second leading cause of cancer-related death in Western men. Although the incidence of prostate cancer was lower than the Western countries', in recent years confirmed PCa, especially for advanced or metastatic PCa, represents an obviously rising trend in China with the aging and the diet habit changing.By2020, the incidence of PCa in China is rising to60-70/100,000. PCa has become one of the threats to men's health in China.
Features such as a high prevalence rate, a long latency period, and complicated pathogenesis have made PCa a desirable tumor to treat. Initially, PCa is androgen-dependent which depends largely on androgen receptor (AR) signaling for growth and maintenance. Androgen deprivation therapy has become the standard first-line treatment for hormone naive PCa, and approximately80%of patients undergo a "flare" response. Unfortunately, the development of hormone-refractory prostate cancer (HRPC) which associated with poor prognosis, high apoptosis-resistance and high mortality will be observed to occur within one or two years after androgen deprivation therapy. There are few efficacious treatment options available for curing, or even improving the survival and quality of life in patients with HRPC. Complicated factors and signals involves in the development of HRPC. Nevertheless, the underlie mechanism of PCa is still remains elusive. Therefore, novel strategies targeting the molecular basis of PCa progression and producing synergies of different signals are highly desirable.We present a large-scale, multi-omic interrogation on the alterations of genes in TCGA datasets, and identified a list of genetic disorders which is associated with malignant phenotypes such as invasion, metastasis, and drug resistance. We then took advantage of the profiles of Chinese Han PCa samples, and found AGR2overexpression was associated with the malignant phenotypes in Chinese Han PCa patients.Further study showed knockdown of AGR2caused DNA damage and resulted incellular senescence in PCa cells.Meanwhile we found natural product bisbibenzyls triggered DNA damage, leading to cellular senescence (at low concentration) and apoptotic cell death (at high concentration), which contributed to its antitumor effect in PCa cells.
Part I Analysis of functional modules of AGR2based on PCa genomic data, and AGR2i-induced DNA damage is involved in regulation of cellular senescence
PCa is the second most common cancer diagnosed in men worldwide. The prevalence of prostate cancer is growing, with an estimated1.1million men worldwide diagnosed in2012, accounting for15percent of the cancers diagnosed in men. In Stage IV prostate cancer, the5-year survival rate is about20%, and the10-year survival is above10%.There are more than200different types of cancer. It has been shown genome-wide genetic changes are correlated with specific types of cancer or the stages of tumor development. It is characterized by a progression of these changes at the cellular, genetic and epigenetic level that ultimately reprogram a cell to undergo uncontrolled cell division, thus forming a malignant mass. The Cancer Genome Atlas (TCGA) is a project to catalogue genetic mutations responsible for cancer, using genome sequencing and bioinformatics. We represented an effort in the War on Cancer that is applying high-throughput genome analysis techniques based on TCGA data to improve our ability to diagnose, treat, and prevent cancer through a better understanding of the genetic basis of this disease.
Anterior gradient protein2homolog (AGR2) is one of candidate therapeutic target genes in PCa arising from the analysis of TCGA data. Current studies showed elevated levels of AGR2in adenocarcinomas of the prostate, breast, lung and pancreas. AGR2is an androgen-inducible protein expressed in normal prostate tissue, and its expression is significantly elevated in PCa tissue compared to benign prostatic epithelial tissue. Urine sediment tests determined AGR2transcript levels to be elevated, and AGR2has been proposed as one of the potential markers to detect malignant cells in the voided urine from PCa patients. In this study, we first observed that AGR2was overexpressed in Chinese Han PCa tissues, and AGR2expression profiles varied among cell lines, with PC3cells being the highest level, LNCaP and DU145relatively less, and hardly detectable in human normal prostate epithelial cell line RWPE1. Here, a potential role of AGR2in cellular senescence was first investigated as well.
一、The genomic landscape of prostate cancer
1. Dysregulation of transcriptome in PCa.
To get a comprehensive overview on transcriptome in PCa, we analyzed RNA sequencing (RNAseq) profiles of256human tumor specimens and45normal specimens from TCGA. We identified24%genes that were up-regulated or down-regulated in the tumor specimen, including28.53%protein-coding gene (PCGene),27.64%long non-coding gene (lncRNA), and16.61%pseudogene.The results indicate complex dysregulation of the cellular transcriptome in PCa.
2.Somatic DNA copy number aberrations in prostate cancer cell genome.
We analyzed the somatic copy number alterations (SCNAs) across258tumor-normal paried samples. Data from high-resolution single nucleotide polymorphisms (SNP) microarray on258PCa specimens and the matching normal specimens were retrieved from TCGA.We found2448genes with high-frequency gain and loss, including961PCGenes,808lncRNAs, and679pseudogenes.
3. Epigenetic analysis of PCa.
To analyze DNA methylation alterations in PCa, we obtained DNA methylation microarray profiles (Infinium Human Methylation450BeadChip) on252human tumor and49normal control specimens from TCGA. We finally identified2617cancer-associated epigenetically silenced genes. The results indicatePCa is characterized by genome-wide methylation imbalance. Next, we performed analysis on miRNA (miRNA-seq) expression profiles of in259human tumor and50normal control specimens from TCGA. We identified36.90%miRNAs that were up-regulated or down-regulated in the tumor specimen compared to normal specimen.
二、Analysis of expression and functional modules of AGR2based on bioinformatic and experimental data.
1. Up-regulation of AGR2in PCa samples.
AGR2is expressed in relatively high levels for prostate cancer patients based on TCGA data. DNA copy number and global methylation analysis showed there were no copy number variations or methylation dysregulation of AGR2in PCa samples. So enhanced mRNA stability and constitutive activation of transcription factor may be involved in upregulation of AGR2.
2. AGR2was overexpressed in Chinese Han PCa tissues.
An elevated expression for AGR2has been observed in prostate tumor tissues of Europe and USA men, however, little is known about the expression level in human tissues among the Chinese Han population. Immunohistochemical analysis of AGR2expression was performed in BPH tissues from41patients, PIN tissues from13patients, and PCa specimens from32patients. The data showed AGR2was overexpressed in Chinese Han PCa tissues as well. We then evaluate the differential expression of AGR2at the protein levels in a panel of various cell lines, and the resluts showedthe expression of AGR2was significantly up-regulated in PCa cells than normal prostate epithelial cells.
3. Analysis of functional modules of AGR2based on TCGA data
we performed Guilt-by-Association (GBA) analysis for AGR2on RNASeq profiles in TCGA to characterize the biological functions and the molecular pathways influenced by AGR2-associated genes. The results showed AGR2correlates with cell cycle, DNA damage and repaired, and cell adhesion.
三、AGR2i-induced DNA damage is involved in regulation of cellular senescence in PCa.
1. Knockdown of AGR2inhibited proliferation in PCa cells.
Knockdown of AGR2by AGR2-targeting siRNA caused a decrease in cell viability in PC3cells, whereas forced expression of AGR2led to an increased cell proliferation of LNCaP and DU145cells.We further assessed the effect of AGR2in cell proliferation process by analysis of incorporation of BrdU into DNA after silencing the endogenous AGR2, and BrdU signaling in AGR2i-treated PCa cells was significantly decreased. Western blotting results showed inactivation of AKT and ERK was accordingly observed in AGR2i-treated cells.
2.Knockdown of AGR2induced cell cycle arrest in PCa cells.
AGR2was overexpressed in Chinese Han PCa tissues and had a positive correlation with Cyclin D1and p-Rb, but not with p16.AGR2-depletion resulted in accumulation of cells at the G0/G1phase.
3. Knockdown of AGR2induced cellular senescence in PCa cells.
AGR2siRNA treatment resulted in cellular senescence in all three PCa cell lines as indicated by an increase of flat, enlarged and senescence-associated β-galactosidase (SA-β-Gal) positive cells. Senescent response to AGR2-silencing was also evidenced by elevated fluorescent punctuate formation of tri-methyl-histone H3in AGR2-depleted cells.
4. AGR2depletion-induced cellular senescence depends on CDKIs.
Further studies indicated that LNCaP underwent a p21-dependent cellular senescence in response to AGR2depletion that requires inactivation of ERK signaling, while PC3was also p21-dependent, but involved in suppression of PI3K/Akt. Unlike LNCaP and PC3, senescent response of DU145was found to be mainly p27-dependent that may require upregulation of PTEN and inhibition of PI3K/Akt signaling.
5. AGR2depletion-induced cellular senescence was accompanied with DNA damage.
Senescence is considered as a DNA damage responseriggered by DNA hyper-replication or other cellular stress. Cells were stained with γH2AX-FITC antibodies and subjected to flow cytometryto quantitatively determine the expression levels of the DNA damage marker γH2AX. Senescent response to AGR2-silencing was evidenced by elevated γH2AX, which means AGR2i-induced DNA damage may be involved in regulation of cellular senescence.
Part Ⅱ DNA damage response indeuced by bisbibenzyls is involved in regulation of cellular senescence and apoptosis in prostate cancer
We found AGR2depletion induced DNA damage and resulted in cellular senescence in PCa cells, and cellular senescence was shown to be one of the key determinants of tumor response to therapy in vitro and in vivo.The concept of pro-senescence therapy has emerged over the past few years as a novel therapeutic approach to treat cancers. Damage to DNA, the prime target of anticancer therapy, triggers programmed cellular responses. In addition to apoptosis, therapy-mediated cellular senescence has been identified as another drug-responsive program that impacts the outcome of cancer therapy.
One of the most efficient chemical reagents used in cancer chemotherapy are DNA damage inducers, which can cause a variety of DNA lesions via multiple mechanisms and resulted in cellular senescence and apoptosis. For example, camptothecin and etoposide can trigger single-strand breaks (SSBs) or double-strand DNA breaks (DSBs) by trapping topoisomerase-DNA covalent complexes, subsequently leading to the cell death; cisplatin and its derivatives forms DNA cross links as monoadduct, interstrand crosslink, intrastrand crosslink or DNA protein crosslink, thus induced DNA damage; cyclophosphamide alkylates the N-7position of guanine and results in frameshift mutation. Cells exposed to chemicals are prone to acquire multiple sites of bulky DNA. Moreover, DNA damaging agents can damage other biomolecules such as proteins, carbohydrates, lipids, and RNA. The accumulation of damage, to be specific, double-strand breaks or adducts stalling the replication forks, are among known stimulation signals for a global response to DNA damage. The global response to damage is an act directed toward the cells' own preservation and triggers multiple pathways of macromolecular repair, lesion bypass, tolerance, senescence, or apoptosis. The common features of global response are induction of multiple genes, cell cycle arrest, and inhibition of cell division.
Depending on the type of DNA lesions, specific cell cycle checkpoints and cellular cascades are activated by DNA-damaging agents. Checkpoint activation pauses the cell cycle and gives the cell time to repair the damage before continuing to divide. As widely accepted, ataxia telangiectasia mutated (ATM) and ataxia telangiectasia and Rad3related (ATR) signaling pathways play important roles in response to DNA damage response. ATM responds mainly to DSBs, and initiates phosphorylation of downstream targets such as Chk2, BRCA1, and NBS1proteins at the site of DNA damage. These factors act together to induce G1, S, and G2cell cycle arrests, DNA repair, and/or activation of cell death pathways. While ATR is activated in response to replication stress, it triggers the activation of Chkl, which in turn leads to the phosphorylation of Cdc25and prevents the activation of CDK1/Cyclin B and mitotic entry. Upon DSBs, the process of DSBs end joining involves numerous proteins and enzymes through nonhomologous end joining (NHEJ) and homologous recombination (HR) repair mechanisms. For example, the Ku70/86heterodimer is critical in NHEJ, since it binds to the broken DNA ends and recruits repair-related proteins including DNA-dependent protein kinase, XRCC4, and DNA Ligase Ⅳ. The rate of DNA repair is dependent on many factors, including the cell type, the age of the cell, and the extracellular environment. A cell that has accumulated a large amount of DNA damage, or one that no longer effectively repairs damage incurred to its DNA, can enter one of three possible states:an irreversible state of dormancy, known as senescence, or cell suicide, also known as apoptosis.
一、Low concentrations of RD triggered p21dependent cellular senescence.
1. Low concentrations of RD induced cellular senescence.
0.5μmole/L RD treatment caused growth arrest at G0/G1phase with features of cellular senescence phenotype such as enlarged and flattened morphology, increased senescence-associated-beta-galactosidase staining cells, and decreased cell proliferation in PCa cells. Induction of cellular senescence by RD occurred through activation of DNA damage response including increases in the γH2AX,inactivation of Chk1/2, and suppression of repair-related Ku70/86and phosphor-BRCA1in PCa cells.
2. RD-triggered cellular senescence is p21denpendent.
It has been demonstrated that the p21-Rb signaling pathway, activated by DNA damage response, plays an important role in the activation of G0/G1checkpoint response and cellular senescence.We then detected the activation of p21in PCa cells, and found the expression of the p21protein was markedly enhanced. We next explored a link between the enhanced p21 expression and senescence induction in response to RD by genetic knockdown of p21. The results revealed that deletion of p21by targeting siRNA impaired RD-induced senescent cells in all three PCa cells, indicating that RD-induced cellular senescence was, at least in part, in a p21-dependent manner.
3. RD triggers cellular senescence in PC3xenograft.
To determine whether RD could induce tumor cell senescence in vivo, as observed in cultured cells, human PC3xenografts were developed in male nude mice. Administration of RD had no effect on body weight but significantly reduced tumor growth of tumor-bearing mice.To examine whether decreases in tumor mass and size by RD were associated with DNA damage-mediated cellular senescence, tissue samples were prepared for immunohistochemical analysis. Consistent with results in culture cells, senescent cells were pronounced in RD-treated tumor tissues compared to the placebo treatment, as evidenced by increased SA-P-Gal staining, associated with substantial accumulation of ting nand positive stained tri-Me-K9-H3which serve as a senescence-associated heterochromatic foci. Meanwhile, RD-mediated alterations in the expressions of p21were similar to the observations shown in culture cells. These data demonstrated that RD triggered DNA damage and cellular senescence that contributed to its observed antitumor effect.
二、RD triggered DNA damage and inhibited DNA repair in PCa cells.
1. RD caused remarkable DNA damage in PC3cells.
After24h treatment with10μmol/L RD, cell cycle was significantly arrested in G2/M phase in PC3cells. In addition to interference with cell cycle progression, RD was able to noticeably induce apoptosis as well. As evidenced by induction of yH2AX foci, micronuclei, and DNA fragmentation in Comet assay, we found RD caused remarkable DNA damage in PC3cells. Time kinetic and dose-dependent studies showed that ATM/Chk2and ATR/Chkl signaling pathways were sequentially activated in response to RD. Blockage of ATM/ATR signaling led to the attenuation of RD-inducedyH2AX, and to the partial recovery of cell proliferation.
2. RD inhibited DNA repair in PC-3cells
Western blotting data showed RD exposure resulted in the inactivation of BRCA1which is a critical molecule in the initial recruitment of other repair proteins/enzymes at the DNA breaks. To determine the effects of RD on DSBs repair, the ability of DNA repair was evaluated with HR and NHEJ reporters. Positive signal significantly declined in either NHEJ or HR repair systems in cells treated with RD, indicating that DSBs repair was impaired in response to RD. Based on the observations above, we further clarified the role of Ku70/Ku86in response to RD-induced DNA damage. DNA end-binding activity of Ku70/Ku86displayed that, compared with the untreated cells, the binding activities of both Ku70/Ku86in treated cells were significantly decreased after12h treatment. Microarray analysis revealed that genes related to damaged-DNA binding, DNA repair, cell cycle, and apoptosis were significantly changed in RD-treated cells.
3. RD triggers apoptosis associated with the induction of DNA damage in PC3xenograft.
Administration of RD to xenograft mice reduced tumor growth, and coordinately caused alterations in the expression of genes involved in DNA damage and repair, along with cell apoptosis.Changes of molecular markers associated with DNA damage in treated mice displayed that RD caused DNA-damage response via ATM/ATR-mediated signaling as evidenced by substantial accumulation of yH2AX, abrogation of phosphor-BRCAl, and Ku70/Ku86abundance. The data demonstrated that RD triggered DNA damage and impaired repair proteins, leading to apoptotic cell death, which contributed to its antitumor effect.
Part III The molecular mechanism of Bisbibenzyls-induced apoptosis in androgen dependent prostate cancer cells
In the last half of the century, advances in tumor cell biology and molecular biology of cancer have been responsible for achievements in cancer therapy. Currently, docetaxel is as an active agent to moderately improve quality of life and survival conditions in patients with metastatic HRPC. However, only about50%of patients treated with docetaxel have a prostate-specific antigen response. Hence, many efforts have been made to investigate novel mechanisms of action of bioactive chemicals with less toxicity and high efficacy to treat HRPC. Macrocyclic bisbibenzyls are a series of phenolic natural products isolated from liverwort, which exert a variety of biological activities, including antifung, antioxidation and cytotoxicity. Our previous research demonstrates its inhibitory effect on PCa cells.
Currently, PI3K pathway is considered a key regulator of progression to androgen-independent prostate cancer, while in androgen-dependent prostate cancer androgen receptor (AR) signaling pathway plays key role in maintaining development, proliferation and differentiation in prostate gland. AR, a nuclear receptor, dissociated from heat shock protein when it bind its ligand, which in turn promotes AR nuclear location and dimerization. Activated AR binds to AR response elements (AREs) and regulates gene transcription, such as PSA. Previous report displayed that AR promoted development from hormone-dependent prostate cancer to horomone-independent prostate cancer. Abnormal androgen signaling due to aberrant expression mutations, or dysregulation of the AR gene has been linked to prostate tumorigenesis, and progression of prostate cancer into advanced, castration-resistant disease. Treatment of androgen-dependent LNCaP cells with macrocyclic bisbibenzyl compound Riccardin C (RC), Marchantin M (MM) or Riccardin D (RD) caused significantly cell cycle arrest and apoptosis. AR is a key factor for cell proliferation in LNCaP cells, so we investigated whether bisbibenzyl compound involved in inhibiting AR function and triggering apoptosis in LNCaP cell.
一、Macrocyclic bisbibenzyl compounds induced apoptosis by inhibiting AR expression and function in LNCaP cells.
1. Exposure of androgen-denpendent cell line LNCaP to RC, MM, or RD caused markedly suppression of cell proliferation.
Treatment of LNCaP cells with RC, MM or RD caused cell cycle arrest in G0/G1phase associated with moderate increases of p53, and p21protein levels, and significantly triggered apoptosis with induction of a higher ratio of Bax/Bcl-2, and elevated cleavage of PARP.
2. Induction of apoptosis was accompanied with the decreased expression and transcriptional activity of the AR.
As demonstrated by western-blotting analysis, AR expression was significantly repressed in LNCaP cells exposed to RC, MM and RD. Luciferase activity assays showed RC, MM and RD treatment markedly suppressed androgen-mediated expression of the AR promoter activity. We further examined the impact of bisbibenzyls on AR transcriptional activity by monitoring the activity of the promoter of prostate specificantigen (PSA), a well-known AR target gene, and found PSA promoter was dramatically repressed by RC, MM and RD in LNCaP cells. We then examined SRC-1/AR interactions using co-immunoprecipitation (co-IP), and declined SRC-1proteins were noticeably observed in cells treated with RC, MM and RD.Thus, inhibitory effect of bisbibenzylson cell proliferation was associated with the downregulation of AR expression,leading to the abolishment of AR.
3. RC, MM and RD targeting proteasome activity promotes autophagy inLNCaP cells.
Proteasome activity assays with purified proteasome in the presence of bisbibenzyls were performed, and the results revealed that RC, MM and RDsignificantly inhibited the proteasome. Werstern blotting and co-IP results demonstrated that the levels of polyubiquitinated-AR were noticeably increased in LNCaP cells treated with RC, MM or RD. We hypothesized that bisbibenzyl-mediated proteasome inhibition simultaneously activates autophagy in LNCaP cells, which in turn is involved in degrading the polyubiquitinated-AR. In our study, induction of LC3B expression and conversion was found in LNCaP cells treated with the compounds which demonstrated the ability of autophagy induction by these agents. We then performed immunofluorescence co-location analysis on AR and LC3or Ub, and the results showed a co-localization of AR and LC3or Ub, suggesting that activation of autophagy by RC, MM and RD might be involved in the process of ubiquitinated ARdegradation.
Part IV Conclusions and Innovation
一、Conclusions
1. A map of prostate cancer genome variation revealed characteristic differences between normal prostate and prostate cancer.
2. AGR2overexpressed in PCa, and knockdown of AGR2caused cellular senescence in PCa cells.
4. Riccardin D induced DNA damage-mediated cellular senescence and apoptosis in prostate cancer.
5. Macrocyclic bisbibenzyl compounds induced apoptosis by inhibiting AR expression and function in androgen-denpendent cell line LNCaP.
二、Innovation
1. With the multi-omic profiles and well-annotated clinical and pathological information on large-scaled clinical samples, we presented the genomic landscape of PCa and provided a powerful resource to improve our ability to diagnose, treat, and prevent cancer through a better understanding of the genetic basis of this disease.
2. We firstly reported AGR2was overexpressed in Chinese Han PCa tissues, and knockdown of AGR2caused cellular senescence in PCa cells.
3. We firstly reported macrocyclic bisbibenzyl compounds prevented AR expression and function, and promoted apoptosis in LNCaP cells. Since AR in LNCaP cells was mutant, the characteristics of bisbibenzyl will be beneficial for the treatment of prostate cancer expression constitutive activation of the AR.
4. We firstly reported RD triggered DNA damage and inhibited DNA repair, thus induced cellular senescence or apoptosis in prostate cancer. Significantly, DNA repair contributes to the drug-resistant phenotype, so that RD may reverse the drug-resistant phenotype by inhibiting repair of chemotherapy-induced genotoxic damage. This finding provides the foundation for the use of RD as a chemotheraputic candidate.
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