【作者】 韩晓蓉；

【导师】 黄宗海；

【作者基本信息】 南方医科大学 ， 普通外科学（专业学位）， 2014， 博士

【摘要】 研究背景和目的乳腺癌是女性最常见的恶性肿瘤之一。据广州市疾病控制中心的统计,2012年乳腺癌发病率已居于广州市城市妇女恶性肿瘤的第一位。乳腺癌的治疗目前仍是以手术为主的包括化疗,内分泌治疗、放疗和分子靶向治疗的综合治疗。乳腺癌是一种异质性疾病,根据其激素受体状态及人上皮细胞生长因子受体2(Her2)的表达将其分为不同的亚型,也常常用这些分子标记物来预测其对靶向治疗的反应性。而随着乳腺癌发生及发展机制研究的深入开展,越来越多的基于乳腺癌发生发展的分子机制的靶向药物被发现,如针对HER-2的曲妥珠单抗治疗,大大改善了乳腺癌患者的预后。然而,我们也看到,仍有相当部分的乳腺癌患者出现了复发和转移,由于肿瘤对化疗、内分泌治疗及靶向治疗药物产生了耐药,从而导致治疗失败。因此,对肿瘤耐药及其机制进行研究,有助于克服耐药,提高疗效。大量研究表明,乳腺癌发生及发展过程中涉及许多信号通路,其中PI3K/Akt/mTOR通路在乳腺癌中存在高频率失调,最高可达70%。在乳腺癌中,PI3K/Akt/mTOR通路的激活在促进乳腺癌细胞的生长,抑制癌细胞凋亡,对化疗及内分泌治疗耐药中起着重要作用,并且与肿瘤分期晚,组织学分级高及预后差密切相关。程序性细胞死亡4(programmed cell death4, PDCD4)是近年新发现的一种肿瘤抑制基因,目前已经发现PDCD4基因在包括乳腺癌在内的多种肿瘤组织及肿瘤细胞中表达下调或缺失。Jansen等通过western-blot方法对美国癌症研究所提供的来自全身10个不同部位共60株人肿瘤细胞株进行PDCD4水平检测,其中在乳腺癌细胞株中,发现恶性程度较高的人乳腺癌MDA-MB-231细胞中的PDCD4表达下调的程度明显高于MCF-7细胞。Wen等比较了正常乳腺组织、导管原位癌(DCIS)及浸润性导管癌中PDCD4的表达情况,发现与正常乳腺组织相比,DCIS中PDCD4表达水平轻度下调,而浸润性导管癌中PDCD4明显下调,说明PDCD4在调节乳腺癌的进展过程中具有重要作用。PDCD4的表达缺失或者下调可能参与恶性肿瘤进展的过程,并维持其高度侵袭的特性,是独立的肿瘤预后因子。目前关于PDCD4的抑癌机制尚不明确,除了可以诱导细胞凋亡,研究提示PDCD4主要起着翻译抑制因子的作用,通过与真核细胞翻译起始复合物中的eIF4A等直接结合,封闭eIF4A的解旋酶活性；以及eIF4A与eIF4G的结合而抑制与细胞增殖、侵袭有关的转录因子的合成,从而发挥其抗肿瘤效应,另有研究表明PDCD4可以促进TIMP-2(Tissue Inhibitor of Metalloproteinases-2,一种抑癌基因)的表达,从而抑制人乳腺癌MCF-7细胞的侵袭能力。PDCD4还可以诱导p21Wafl/Cipl水平升高后减少CDK4/6和CDK2引起细胞周期阻滞,从而减少细胞增殖。根据现有文献报导,miR-21、TGF-β、mTOR、COX-2等多种因子都参与了PDCD4的调控过程。其中mTOR/P70S6k信号通路在PDCD4表达的调控中起到重要作用。Dorrello等研究发现,在促细胞分裂素的刺激下,细胞内的S6K1被激活,活化的S6K1(即P70S6k1)使PDCD4在Ser67处迅速磷酸化,随后磷酸化的PDCD4与泛素连接酶SCFβTRCP结合被泛素化,最后被蛋白酶体降解。而Ser突变的PDCD4变异体却不能磷酸化。作为S6K1的同源体,S6K2也被证实可以促进PDCD4的磷酸化降解。由于PDCD4含有两个Akt磷酸化位点Ser67和Ser457,且在体内和体外Akt能够特异地磷酸化PDCD4的Ser67和Ser457残基,因此Akt也被认为直接参与了PDCD4的降解。由于S6k1的活性主要受上游细胞信号通路PI3k/Akt/mTOR的调节,因此任何引起其上游信号活性改变的因子均可通过影响S6kl的活性而间接调控PDCD4的表达。S6K1是mTORC1下游的靶点之一,雷帕霉素治疗可以迅速使S6K1去磷酸化而失活。在mTORC1/S6K1信号通路与ER信号之间存在着密切的关联,ER阳性乳腺癌对内分泌治疗耐药与nTOR信号通路的过度活化引起的不依赖配体的ERa信号的活化相关。S6K1可以直接在Ser167激活ERa,从而导致ERa转录活性增加,促进ER依赖的乳腺癌细胞增殖,而反过来,ERa的活化又导致S6K1表达的增加,形成正向共调节回路。有研究表明P-S6K1在ER阳性乳腺癌中与不良预后相关。S6K1的过表达可提高乳腺癌细胞对雷帕霉素的敏感性。S6K2是S6K1的同源蛋白,也是mTORC1下游的靶点,在乳腺癌中也有过表达。有研究表明S6K2在小细胞肺癌FGF2诱导的化疗耐药中起作用,并且在应答mTOR活化的细胞增殖中起着重要作用。S6K2还可以通过使Akt磷酸化而促进乳腺癌细胞的生长。雷帕霉素也称西罗莫司(sirolimus),是发现的第一个mTOR抑制剂。在细胞内,雷帕霉素首先与FKBP12结合形成复合物,然后再与mTORC1结合,抑制其活性,使细胞周期停滞在G1期,并促进细胞凋亡。临床上已将雷帕霉素及其类似物用于治疗各种实体肿瘤,包括乳腺癌,它可以提高肿瘤细胞对化疗及内分泌治疗的敏感性。但是在实际应用中却发现雷帕霉素的治疗效果有限,且易产生耐药性。其耐药性产生的机制目前尚不十分清楚,可能与RAS/MEK/ERK及TGF-B/CTGF信号通路的激活有关。根据文献报道,MCF-7细胞及MDA-MB-231细胞中均存在mTOR异常激活,两者中Phosho-mTOR水平基本一致,并且两者中均未检测到phosho-Akt,差别在于MCF-7细胞中phosho-S6K1水平明显高于MDA-MB-231细胞,而MDA-MB-231细胞中PDCD4表达的下调较MCF-7明显。另所文献报道,MDA-MB-231细胞对雷帕霉素原发耐药,MCF-7细胞则可对雷帕霉素产生获得性耐药。基于上述研究结果,我们假设PDCD4可能在雷帕霉素治疗乳腺癌耐药中起作用。本研究发现,与正常乳腺组织相比,PDCD4在乳腺癌组织中有不同程度的表达缺失,在不同分子亚型的乳腺癌中缺失率不同,在三阴性乳腺癌和HER2阳性乳腺癌中其表达缺失率明显高于管腔型乳腺癌,且其表达缺失与组织学分级高,淋巴结转移相关,PDCD4表达阳性患者的无病生存及总生存均优于表达阴性者。P70S6K在乳腺癌组织中的表达水平明显高于正常乳腺组织,其表达阳性者无病生存及总生存均明显差于表达阴性者。本研究还发现,雷帕霉素可以上调MDA-MB-231细胞中PDCD4的表达水平,其上调是通过使S6K1及S6K2去磷酸化而实现的,PDCD4表达上调后其细胞增殖受到抑制。雷帕霉素对MCF-7细胞中PDCD4的表达影响与MDA-MB-231细胞完全不同,是先上调而后下调,而这种表达的影响是通过S6K2的磷酸化来实现的,PDCD4的下调导致了细胞增殖的增强。雷帕霉素对三阴性乳腺癌细胞MDA-MB-231和ER阳性乳腺癌细胞MCF-7中PDCD4表达的影响完全不同,可能是其对雷帕霉素治疗产生耐药的原因之一。因此,本研究为进一步研究雷帕霉素治疗乳腺癌过程中耐药性产生的机制提供了新的实验依据,并为克服这种耐药性提供了可能。本研究分为二部分,摘要如下：第一部分PDCD4及p70S6K在不同乳腺癌组织中的表达及意义目的：研究不同分子亚型乳腺癌组织中PDCD4及p70S6K的表达及其与乳腺癌临床病理特征和预后的关系方法：1.采用免疫组化SP法分别检测不同分子亚型乳腺癌组织中PDCD4及P70S6K的表达,其中Luminal型乳腺癌198例,HER2阳性型64例及三阴性乳腺癌76例。2.比较不同分子亚型乳腺癌组织中PDCD4及P70S6K的表达水平与乳腺癌临床病理特征的关系。3.采用Kaplan-Meier法对各组患者的无病生存及总生存情况进行分析,采用Log-rank检验比较生存曲线间的差异。采用Cox回归模型对各组患者的无病生存及总生存情况进行多因素分析,找出影响预后的因素。结果：1.PDCD4蛋白在不同分子亚型乳腺癌组织中的表达比较：所有乳腺癌组织中PDCD4蛋白总阳性表达率为58.28%,表达缺失率为41.72%。其中管腔型、Her-2阳性及三阴性乳腺癌组织中,PDCD4蛋白的阳性表达率分别为74.36%、33.33%及36.84%,表达缺失率分别为25.64%、66.67%及63.16%。三种分子亚型乳腺癌中PDCD4的阳性表达率之间差异有统计学意义(χ2=51.685,P<0.001)；其中管腔型乳腺癌中PDCD4的阳性表达率高于Her2阳性及三阴性乳腺癌,差异有统计学意义(χ2=35.197,P<0.001,χ2=33.620,P<0.001),而Her2阳性及三阴性乳腺癌中PDCD4的阳性表达率之间差异无统计学意义(χ2=0.186,P=0.666)。癌旁正常乳腺组织中PDCD4蛋白阳性表达率为96.44%。三个亚型乳腺癌组织中PDCD4的阳性表达率均低于癌旁正常组织中,差异有统计学意义(χ2=39.206,P<0.001；χ2=60.398,P<0.001；χ2=65.185,P<0.001)。2.不同分子亚型乳腺癌组织中p70S6k的表达：正常乳腺组织中不表达p70S6k。所有乳腺癌组织中,p70S6k的阳性表达率为23.96%,其中管腔型、Her-2阳性及三阴性乳腺癌组织中,p70S6k蛋白的阳性表达率分别为12.06%、38.09%及36.84%,各型乳腺癌组织中p70S6k的阳性表达率之间差异有统计学意义(χ2=30.204,P<0.001)。Her2阳性及三阴性乳腺癌中p70S6k的阳性表达率高于管腔型乳腺癌,差异有统计学意义(χ2=21.675,P<0.001,χ2=22.027,P<0.001),Her2阳性及三阴性乳腺癌中p70S6k的阳性表达率之间差异无统计学意义(χ2=0.023,P=0.879),。3.PDCD4表达对不同分子分型乳腺癌预后的影响：单因素分析结果显示,338例乳腺癌中PDCD4表达阳性者其无病生存及总生存均明显优于表达阴性者(χ2=71.403,P<0.001；χ2=34.919,P<0.001)；而在各分子亚型乳腺癌中,同样显示出PDCD4表达阳性者其无病生存及总生存明显优于表达阴性者。多因素分析显示,影响乳腺癌无病生存的独立因素包括肿瘤T分期,N分期及PDCD4表达(P<0.01,P<0.01,P<0.001)；影响乳腺癌总生存的独立因素包括组织学分级,肿瘤T分期,N分期及PDCD4表达(P<0.01,P<0.01,P<0.001,P<0.01)结论：1.乳腺癌组织中PDCD4蛋白表达呈现不同程度的缺失,其中Her2阳性及三阴性乳腺癌中PDCD4蛋白表达缺失最明显。2.乳腺癌组织中P30S6K蛋白表达较正常乳腺组织明显增多,其中Her2阳性及三阴性乳腺癌中P70S6K蛋白表达增多最明显。3.乳腺癌组织中PDCD4蛋白表达缺失与组织学分级高,淋巴结转移相关。4.无论何种分子亚型,PDCD4表达阳性的乳腺癌其无病生存及总生存均优于表达阴性者。第二部分雷帕霉素刺激对三阴性乳腺癌细胞株MDA-MB-231及ER阳性乳腺癌细胞株MCF-7中PDCD4表达的影响及机制目的：研究雷帕霉素刺激后三阴性乳腺癌细胞株MDA-MB-231及ER阳性乳腺癌细胞株MCF-7中PDCD4表达的变化及意义方法：1. MDA-MB-231细胞及MCF-7株培养传代后,100nM雷帕霉素处理细胞株2.分别在处理后24小时及48小时,western blot分析雷帕霉素对两个细胞株中PDCD4表达水平的影响3.分别在处理后24小时及48小时,western blot检测两个细胞株中P70S6K1及P70S6K2的表达变化,研究雷帕霉素影响两个细胞中PDCD4表达的机制4.雷帕霉素刺激两种细胞株后,检测细胞增殖情况,研究雷帕霉素影响PDCD4表达的意义5.所有数据采用SPSS17.0软件包进行统计学处理,western-blot结果记录为均数±标准差,采用单样本t检验或独立样本t检验进行统计学分析,P<0.05为差异有统计学意义。结果：1.雷帕霉素100nM分别孵育MDA-MB-231细胞24h、48h、Western blot检测PDCD4在MDA-MB-231细胞中的表达,结果显示雷帕霉素孵育细胞24h、48h后均上调了MDA-MB-231中PDCD4的表达。(P<0.05,n=3,VS0h组)；雷帕霉素100nM分别刺激MCF-7细胞24h、48h、Western blot检测PDCD4在MCF-7细胞中的表达,结果显示雷帕霉素孵育细胞24h后也上调了PDCD4的表达。但雷帕霉素孵育细胞48h后,MCF-7中PDCD4的表达却显著下调了(P<0.01,n=3,VS0h组)2.雷帕霉素100nM孵育MDA-MB-231细胞24h, Western blot检测PDCD4、p-S6K1及p-S6K2在MDA-MB-231细胞中的表达,结果显示雷帕霉素孵育细胞24h后,在PDCD4表达上调的同时,p-S6K1及p-S6K2的表达均下调。(P<0.01,n=3, VS control);雷帕霉素100nM孵育MCF-7细胞24h, Western blot检测PDCD4、p-S6K1及p-S6K2在细胞中的表达,结果显示雷帕霉素孵育细胞24h后,p-S6K1的表达下调了(P<0.05, n=3, VS control), p-S6K2的表达与空白对照及DMSO组比较差异无统计学意义(P=0.839, n=3, VS control)3.雷帕霉素100nM孵育MDA-MB-231细胞48h, Western blot检测PDCD4、p-S6K1及p-S6K2在MDA-MB-231细胞中的表达,结果显示在PDCD4表达上调的同时,同样p-S6K1及p-S6K2的表达均下调了,以p-S6K1更为显著。(P<0.01, n=3, VS control);雷帕霉素100nM孵育MCF-7细胞48h, Western blot检测PDCD4. p-S6K1及p-S6K2在MCF-7细胞中的表达,结果显示在PDCD4表达显著下调的同时,p-S6K2的表达上调(P<0.01, n=3, VS control), p-S6K1的表达与对照组比较差异无统计学意义(P=0.769, n=3, VS control)。4.雷帕霉素刺激MDA-MB-231细胞株后,随着刺激时间延长,细胞增殖被明显抑制；雷帕霉素刺激MCF-7细胞株后,随着刺激时间延长,细胞增殖在24h被抑制,其后细胞增殖呈现增强趋势。结论：1.雷帕霉素刺激可上调MDA-MB-231细胞株中PDCD4的表达,其上调PDCD4表达可能是通过下调P-S6K1及P-S6K2的表达实现的；2.雷帕霉素刺激先上调后下调MCF-7细胞株中PDCD4的表达,雷帕霉素对MCF-7细胞株中PDCD4表达的影响与MDA-MB-231细胞株不同,在刺激早期是由P-S6K1下调实现的,后期则是通过过上调P-S6K2的表达实现的3.雷帕霉素上调MDA-MB-231细胞株中PDCD4表达后,可抑制其细胞增殖,表明PDCD4表达上调可能解除了MDA-MB-231细胞株对雷帕霉素的耐药性；雷帕霉素先上调后下调MCF-7细胞株中PDCD4表达后,其细胞增殖先受抑制其后抑制解除,表明PDCD4表达下调可能导致了MCF-7细胞株对雷帕霉素产生获得性耐药4.乳腺癌细胞中PDCD4表达的下调在乳腺癌对雷帕霉素耐药中起作用。更多还原

【Abstract】 Background and ObjectiveBreast cancer is one of the most common malignancies in women. According to the statistics of Guangzhou Center for Disease Control, breast cancer incidence ranks the first place of women malignancy in Guangzhou, in2012. Breast cancer is a heterogeneous disease. According to the hormone receptor expression status and epithelial cell growth factorreceptor2(Her2), it is classified as different subtypes, and these molecules are often used to predict the therapy responses.With the development of breast cancer research, more targeted drugs based on molecular mechanisms was found, such as trastuzumab which is targeted to HER2, and this target therapy has greatly improved the prognosis of breast cancer. However, there is still a considerable part of breast cancer recurrence and metastasis. Because the tumor is resistance to chemotherapy, endocrine therapy and targeted therapy. Therefore, we need to study its mechanism of tumor resistance so that we can overcome drug resistance and improve efficacy.Numerous studies have shown that there are many signaling pathways involved in the development ofbreast cancer, including the presence of high frequency offset of PI3K/Akt/mTORpathway which is up to70%. PI3K/Akt/mTORpathwayactivati onpromotethe growth ofbreast cancer cells, inhibit cancer cell death, and play an important role in the process of chemo-andendocrine-therapy resistance. It is also closely related with thetumor stage, histological grade andpoor prognosis.Programmed cell death4(PDCD4)wasrecently discoveredas a tumorsuppressor gene. Its downregulationhas now been foundinmany tumor tissuesand tumorcel lsincluding breast cancer. Jansen et al. detected PDCD4levels in60human tumor cell lines provided by the American Institute for Cancer Research by western-blot. Those cell lines are from10different parts of the body. In breast cancer cell lines, they found that PDCD4level was significantly downregulated in MDA-MB-231cell linescompared with MCF-7cells.Wen et al. detected the expression ofPDCD4in those relativelynormalbreast tissue, ductal carcinoma in situ(DCIS)andinvasive ductal carcinomaand theyfound that compared withnormal breast tissue, the expression level ofPDCD4in DCIS slightlydecreased, and get significantly decreased in invasive ductalcarcinoma, indicating an important rolein the regulation ofbreast cancerprogression. Missing orreducedexpression of PDCD4may be involved inthe process oftumorprogressionand maintain highlyinvasivecharacteristics. Thisis an independentprognostic factor forcancer. Currently the mechanism of PDCD4as a tumor suppressor gene is not very clear. Besides apoptosis induction, studies have suggested thatPDCD4plays amajorrole intranslational inhibition, with theeukaryotic translationinitiation complex, such as direct bindingofeIF4A, eIF4A’s closedhelicase activity; and the combination ofeIF4AwitheIF4Ginhibitcell proliferation,invasion andsynthesisoftranscription factors, which play itsanti-tumor effect. Another studyshowed thatPDCD4can promoteTIMP-2(Tissue Inhibitor of Metallopro teinases-2,a tumor suppressor gene) expression, thereby inhibit the invasive ability ofhuman breast cancerMCF-7cell line. PDCD4can alsoinducep21Wafl/Ciplex pressionafterreducingCDK4/6andCDK2, then cause cellcycle arrest, thereby stopping cell proliferation.According toliteraturereports, miR-21, TGF-β, mTOR, COX-2, and other factorsare involved intheregulation ofPDCD4. And mTOR/P70S6ksignaling pathwayplays an important rolein the regulation ofthe expressionofPDCD4. Dorrello et al. have foundthat S6K1can be activated by mitogenstimulation. The activationofS6Kl (ie P70S6k1) lead to phosphorylation of PDCD4atSer67siterapidly, followed by the binding of phosphorylatedPDCD4to ubiquitinligase, then they areubiquitinated, and finally degraded by the proteasome. ButSermut antvariantsPDCD4could not bephosphorylated.As ahomolog ofS6Kl, S6K2has also beenshown topromote thedegradation ofPDCD4by phosphorylation. SincePDCD4containstwositesof phosphorylation of AktSer67andSer457,and it is capable of specificallyphosphorylatedatSer67andSer457by Akt both in vivo and in vitro,Aktis also consideredto be involvedin PDCD4degradation directly. SinceS6klactivityis mainly affected bythe upstreamregulatorof cell signalingpathwaysPI3k/Akt/mTOR, so anyfactorcausingthe upstreamsignaling activitycan bealteredby affectingthe activity ofS6kl and thus indirectlyregulatethe expression ofPDCD4.S6Klis one of thedownstreamtargets ofmTORC1, rapamycin treatmentcan quicklymakeS6Kldephosphorylationandinactivation.BetweenmTORC1/S6K1and ERsignalpathway, there is a closeassociation:the endocrine therapy resistance of ER-positive breast cancer is associatedwiththe activation ofmTORsignaling pathway.S6K1can directly activateERa in Ser167site,resulting in increased transcription activity ofERa andpromotingER-dependent breast cancer cell proliferation. And ERa activationin turn leads toincreasedexpressionofS6Kl, then the positiveregulatorcircuitis formed. Studies have shown thatP-S6KlinER-positive breast cancer is associated withpoor prognosis.Overexpression ofS6K1can increa sethe sensitivity ofbreast cancercells torapamycin. S6K2is the homologousprotein of S6Kland is the downstreamtargets of mTORC1,it is alsooverexpressed in breast cancer. Studies have shown thatS6K2played a role in chemotherapy resistanceof small cell lung cancerinduced byFGF2andit also plays an importantrole in cell proliferationinresponseto mTORactivation. S6K2can alsomakeAktphosphorylationan dpromote the growth ofbreast cancer cellsRapamycin, also known assirolimus,is the firstmTORinhibitor.Inside the cell, rapamycin-FKBP12bindingcomplexesformed at the beginning, and then combinedwithmTORCl,inhibiting its activity, making cell cyclearrest inGlphase, then inducingapoptosis.Clinically,rapamycinand its analogshave beenused for the treatmentof solid tumors, including breast breast cancer. It canimprove the sensitivityof tumorcells tochemotherapy andendocrine therapy. Butin practice the Iimitedtherapeutic effectof rapamycin was verified, and it is also easy to producedrug resistance. However, the mechanismsofdrug resistanceis not very clear. Possibly, RAS/MEK/ERKsignaling pathwayandTGF-B/CTGF pathway arerelevant. According to literature, abnormal activation ofmTORwere present in both MCF-7cells andMDA-MB-231cells, and the level of Phosho-mTORwere basically the same, and phosho-Akt wereb not detectedin the two cell lines. The differencelies inthat the level of phosho-S6Klwere significantly higher in MCF-7cell lines thanthat in MDA-MB-231cell lines, andthe expresstion ofPDCD4was down regultated significantly in MDA-MB-231cell linescompared withMCF-7cell lines. Alsoasreported in other studies, MDA-MB-231cells are primarily resistant torapamycin; MCF-7cellscan begeneratedforacquired resistance torapamycin. Based on these findings, we assume thatPDCD4mayplay a role inthe resistance to rapamycin during the treatment ofbreast cancer.In our study, we found that, there arevarious degrees oflossof PDCD4expression in breast cancercompared with normalbreast tissue,the loss is also variant in different molecular subtypes. Intriple negative breast cancerand HER2-positive breast cancer,its expressiondeletion ratewas significantly higher thanluminal types, and is associated withhighhistological grade,lymph nodemetastasis.Patientswith PDCD4expression have higherdisease-freesurvival and overall survival rates. We also found thatrapamycincanincreasethe expression level ofPDCD4in MDA-MB-231cells. This process is achieved byS6K1andS6K2dephosphorylation. After PDCD4upregulation, cell proliferationwas inhibited. The effects of Rapamycin on PDCD4expression inMCF-7cells are totally different from MDA-MB-231, andPDCD4firstlyin creasesand thendecreases, andthe effectis throughphosphorylation ofS6K2. Downregulationof PDCD4resulted inenhancementof cellproliferation in MCF-7. The effects of Rapamycin on theexpressionofPDCD4intriple-negativebreast cancer cellsMDA-MB-231andER-positive breast cancer MCF-7cells are completely different,probably this is one of thereasonsforrapamycinresistance. Therefore, the present studyisto furtherstudythe mechanismof rapamycinresistancein breast cancertreatment, intends to provides anew experimentalbasis, andto overcomethis drug resistance. Ourstudy is divided intotwo parts, which are summarized as follows:Part I:PDCD4andp70S6K expression and their significance in different subtypes of breast cancerObjective: Tostudytheexpressionof PDCD4andp70S6K in different molecularsubtypes ofbreast cancertissuesand its relationship withclinicopathol ogicalfeaturesand prognosisMethods:1.SP immunohistochemistry was usedto show P70S6K and PDCD4expression in differentmolecularsubtypes ofbreast cancer. Samples:198cases ofbreast cancer of Luminal type,64cases of HER2-positive typeand76cases of triple-negativetype.2.Compare expression levels ofPDCD4andP70S6K in differentmolecules subtypes ofbreast cancer and analyze their relationshipwithclinicopathological features.3. UseKaplan-Meier statistic method to analyze the disease-free survival and overallsurvival ofpatients in each group.Log-ranktest is introduced to analyzes urvivalcurvesofdifferent groups. Cox regressionmodel is used for multivariateanalysis of disease-freesurvival and overallsurvival and to identifyprognostic factors.Results:1.PDCD4proteinexpressionindifferent molecularsubtypes ofbreast cancer: in allbreast cancer, the total rate of PDCD4proteinexpression was58.28%, and in luminaltypes, Her-2positiveandtriple negativetypes, PDCD4proteinexpression ratewere74.36%、33.33%及36.84%respectively. The expression rate in Luminal type was significantly higher thanthat in Her2positive andtriple negative types, and the difference was statistically significant (X2=35.197,P<0.001,X2=33.620,P<0.001). In Her2-positive andtriple negative breast cancer, there was no significant difference (X2=0.186, p=0.666) about the expression rate. In adjacent normalbreast tissue,PDCD4protein expression rate was96.44%, much higher than the threesubtypes ofbreast cancerand thedifferencewas statistically significant (X2=39.206, P<0.001; X2=60.398, P <0.001; X2=65.185, P<0.001).2. p70S6k expression indifferent molecularsubtypesofbreast cancer: there was no expressionof p70S6K in normal breast tissue. For allbreast cancer, the expression rate was23.96%, in which the luminaltype, Her-2positiveandtriple negative, the expression rates were12.06%,38.09%and36.84%, respectively. The expression rates inHer2-positive andtriple-negativegroupswere significantly higher thaninluminal group (X2=21.675,P<0.001, X2=22.027,P<0.001). There was no significant differencebetween Her2positive andtriple-negative group (X2=0.023, P=0.879) 3. The effects of PDCD4expression onbreast cancerprognosis in differentmolecular subtypes:Univariate analysisshowed that in338breast cancer cases,PDCD4expression is positively related withdisease-free suuvial and overall survival (X2=71.403, P<0.001; X2=34.919, P<0.001);ineachmolecular subtypeof breast cancer, data also showsPDCD4expression is positively related with disease-free survival and overall survival (X2=50.997, P<0.001).Multivariate analysisshowed thatthe tumorTstage, N stageandPDCD4expression were independentfactors for the disease-free survivalin breast cancer (P<0.01, P<0.001,P <0.001);and histological grade of tumor, Tstage, N stageandPDCD4expression were the independentfactorsfor the overall survival in breast cancer (P<0.01, P<0.01,P <0.001,P<0.01)Conclusions:1.There were various degree of loss of PDCD4protein expressionin breast cancer, whichis the mostobvious in Her2-positive andtriple-negativebreast cancer2.P70S6K expressionincreased significantly in breast cancer tissuecompared tonormal breast tissue,whichHer2-positive andtriple-negativebreast cancer P30S6Kincreasedthe mostobviously.3.In breast cancerthe lack of PDCD4expression is associated with high histological gradeand lymph node metastasis.4.Regardless ofmolecular subtypes, patients with positive PDCD4expression have betterdisease-free survival and overall survival.Part Ⅱ The impact of rapamycin on PDCD4expression in triple negative breast cancer cell line MDA-MB-231and ER positive breast cancer cell line MCF-7 Objective:To study the changes of PDCD4expression in triple negative breast cancer cell line MDA-MB-231and ER positive breast cancer cell line MCF-7treated by rapamycinMethods:1. MDA-MB-231and MCF-7cell line grows in complete medium and in its Logarithmic growth phase, cells were treated by100nM rapamycin.2. Cells were harvested after24or48hours treatment, then PDCD4expression was analyzed by western blotting.3. Similarly, P70S6K1and P70S6K2expression were analyzed respectively by western blotting in MDA-MB-231cells and MCF-7cells treated by rapamycin for24or48hours. The mechanism of rapamycin on PDCD4expression in two breast cancer cells was further studied.4. After rapamycin treatment, cell proliferation was determined, and the significance of rapamycin effects on the PDCD4expression was explored.5. All data were statistically analyzed using SPSS17.0software package, western-blot results were recorded as mean±standard deviation, one-sample t test or independent sample t test was used for statistical analysis, P<0.05was considered statistically significant.Results:1.MDA-MB-231cellswereincubated with100nMrapamycin for24h,48h. Western blottingshowed increased PDCD4expression.(P<0.01, n=3, VS Ohgroup);MCF-7cells were treated by100nMrapamycin for24h,48h respectively. Western blotting data showed increased PDCD4expression after24h incubation, but after longer time (48h), PDCD4expression decreased significantly(P<0.01, n=3, VS Ohgroup).2.MDA-MB-231cellswereincubated with100nMrapamycin for24h. Western blotting showed that together with increased PDCD4expression,the expression ofp-S6Klandp-S6K2inMDA-MB-231cells decreased.(P<0.05, P<0.01, P<0.01, n=3, VS control); MCF-7cells were treated by100nMrapamycin for24hs. Western blotting was used to detect PDCD4, p-S6Klandp-S6K2expressionin cells. Data showed that the expression ofp-S6Klislowered, but p-S6K2expression appeared no significant change compared tothe controlandDMSO group.(P=0.839, n=3, VS control).3. MDA-MB-231cellswereincubated with100nMrapamycin for48hs. Western blotting showed that together with increased PDCD4expression,the expression ofp-S6Klandp-S6K2inMDA-MB-231cellsalso decreased.(P<0.05, P<0.01, P<0.01, n=3, VS control); MCF-7cells were treated by100nMrapamycin for48hs. Western blotting was used to detect PDCD4, p-S6Klandp-S6K2expressionin cells. Data showed that the expression ofp-S6K2isupregulated, but p-S6K1expression appeared no significant change compared tothe controlandDMSO group.(P=0.769, n=3, VS control).4. After rapamycin treatment, the proliferation of MDA-MB-231cells was significantly inhibited; MCF-7cell growth was inhibited firstly, but then its proliferation was promoted.Conclusions:1.Rapamycin canincreasePDCD4expression inMDA-MB-231cell lines; Rapamycin treatment firstly up-regulated and then down-regulated PDCD4expression in MCF-7cell lines.2. The up-regulation effects of rapamycin on PDCD4expression in MDA-MB-231cell linesmay be due to the downregulation of both P-S6KlandP-S6K2. The down-regulation effects of rapamycin on PDCD4expression in MCF-7cell lines was also related to P-S6KlandP-S6K2.PDCD4expression was up regulated by P-S6K1in early stage of stimulation by rapamycin and the following downregulation of PDCD4might be achievedby up-regulatingP-S6K2.3.Rapamycin could inhibit the cell proliferation in MDA-MB-231by up regulateing PDCD4expression,and thusconfront the resistance to rapamycin; and in MCF-7cells, the expression of PDCD4was increased firstly and then decreased by rapamycin treatment correspondingly, and the cell growth was inhibited firstly and then disinhibited. These data suggestied that PDCD4downregulationmay result in the rapamycinresistance in MCF-7cell lines.更多还原