雷帕霉素对乳腺癌细胞中程序性细胞死亡4表达的影响及其机制的研究
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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。
Background and Objective
Breast 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 cells
Rapamycin, 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 cancer
Objective: Tostudytheexpressionof PDCD4andp70S6K in different molecularsubtypes ofbreast cancertissuesand its relationship withclinicopathol ogicalfeaturesand prognosis
Methods:
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 cancer
2.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 rapamycin
Methods:
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.
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。
Background and Objective
Breast 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 cells
Rapamycin, 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 cancer
Objective: Tostudytheexpressionof PDCD4andp70S6K in different molecularsubtypes ofbreast cancertissuesand its relationship withclinicopathol ogicalfeaturesand prognosis
Methods:
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 cancer
2.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 rapamycin
Methods:
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.
引文
1.Carvalho S,Schmit F.Potential role of PI3Kinhibitors in the treatment of breast cancerr[J].Future Oncol,2010,6(8):1251-1263
2.Guillermet G J, Bjorklof K, Salpekar A, et al. The p110beta isoform of phosphoinositide 3-kinase signals downstream of G protein-coupled receptors and is functionally redundant with pllOgamma. ProcNatl Acad Sci,2008; 105(24):8292-8297
3. Shaw RJ, Cantley LC. Ras, PI(3)K and mTOR signalling controls tumour cell growth. Nature. 2006; 441(7092):424-430.
4.Skolnik EY, Margolis B, Mohammadi M, et al. Cloning of PI3 kinase-associated p85 utilizing a novel method for expression/cloning of target proteins for receptor tyrosine kinases. Cell. 1991;65(1):83-90.
5.Maehama T, Dixon JE. Thetumorsuppressor,PTEN/MMAC1,dephosphorylates the lipid second messenger, phosphatidylinositol3,4,5-trisphosphate. J Biol Chem. 1998;273(22):13375-13378.
6.Cheng JQ, Lindsley CW, Cheng G.Z, et al. The Akt/PKB pathway:molecular target for cancer drug discovery. Oncogene, 2005; 24(50):7482-7492,
7.Sokunaga E,Oki E, Egashira A,et al.Deregulation of the Akt pathway in human cancer[J].Cur Cancer Drug Targets,2008,8(1):27-36
8.Feng J, Park J, Cron P, et al. Identification of a PKB/Akt hydrophobic motif Ser-473 kinase as DNA-dependent protein kinase. J Biol Chem.2004; 279(39):41189-96.
9.Hresko RC, Mueckler M. mTOR.RICTOR is the Ser473 kinase for Akt/protein kinase B in 3T3-L1 adipocytes. J Biol Chem. 2005; 280(49):40406-16.
10. Wullschleger S, Loewith R, Hall MN. TOR signaling in growth and metabolism. Cell 2006; 124:471-84.
11. Wang L, Harris TE, Lawrence JC, Jr. Regulation of proline-rich Akt substrate of 40 kDa (PRAS40) function by mammalian target of rapamycin complex 1 (mTORCl)-mediated phosphorylation. J Biol Chem 2008; 283:15619-27.
12. MericBF, Gonzalez AM. Targeting the mTOR signaling network for cancer therapy. J Clin Oncol 2009; 27:2278-87.
13.Benedetti A, Graff JR. eIF-4E expression and its role in malignancies and metastases. Oncogene 2004; 23:3189-99
14. Jastrzebski K, Hannan KM, Tchoubrieva EB, et al. Coordinate regulation of ribo-some biogenesis and function by the ribosomal protein S6 kinase, a key mediator of mTOR function. Growth Factors 2007; 25:209-26.
15. Nakamura JL, Garcia E, Pieper RO. S6K1 plays a key role in glial transformation. Cancer Res 2008; 68:6516-23
16. Barlund M, Forozan F, Kononen J, et al. Detecting activation of ribosomal protein S6 kinase by complementary DNA and tissue microarray analysis. J Natl Cancer Inst 2000; 92:1252-9
17. Garc A, Martinez JM,Aleesi DR. mTOR complex 2 (mTORC2) controls hydrophobic motif phosphorylation and activation of serum-and glucocorticoid-induced protein kinase 1 (SGK1)[J]. ChemJ,2008,416(3):375-385.
18. Jacinto E, Facchinetti V, Liu D, et al. SIN1/MIP1 maintains rictor-mTOR com-plex integrity and regulates Akt phosphorylation and substrate specificity. Cell 2006; 127:125-37.
19. Sarbassov DD, Ali SM, Kim DH, et al. Rictor, a novel binding partner of mTOR, defines a rapamycin-insensitive and raptor-independent pathway that regulates the cytoskeleton. Curr Biol 2004; 14:1296-302.
20. Hernandez-Negrete I, Carretero-Ortega J, Rosenfeldt H, et al. P-Rexl links mammalian target of rapamycin signaling to Rac activation and cell migration. J Biol Chem 2007; 282:23708-15
21.Noh EM,Le YR,Chay KO,et al.PTEN and the PI3-kinasepathway in cancer[J].Ann Review Pathol,2009,4(2):127-150
22. Demidenko ZN,Shutman M,Blagosklonnyl MV. Pharmacologic inhibition of MEK and PI-3K converges on the mTOR/S6 pathway to decelerate cellular senescence[J]. Cell Cycle, 2009,8(12):1896-1900
23.JACINTO E,LOEWITH R,SCHMIDT A,et al. Mammalian TOR complex 2 controls the actin cytoskeleton and is rapamycin insensitive [J]. Nat Cell Biol,2004,6(11):1122-1128.
24. Sarbassov DD,Ali SM,Kim DH,et al. Rictor,a novel binding partner of mTOR, defines a rapamycin-insensitive and raptor-independent pathway that regulates the cytoskeleton[J]. Curr Biol,2004,14(14):1296-1302
25. Rosner M,Fuchs C,Siegel N,et al. Functional interaction of mammalian target of rapamycin complexes in regulating mammalian cell size and cell cycle[J]. Hum Mol Genet, 2009,18(17):3298-3310.
26. Yu Y,Yoon SO,Poulogiannis G,et al. Phosphoproteomic analysis identifies Grb10 as an mTORC1 substrate that negatively regulates insulin signaling[J]. Science,2011,332 (6035):1322-1326.
27. Agarwal R, Carey M, Hennessy B, et al. PI3K pathway-directed therapeutic strategies in cancer. Curr Opin Investig Drugs 2010; 11:615-28.
28. Foster KG,Fingar DC. Mammalian target of rapamycin (mTOR):conducting the cellular signaling symphony[J]. J Biol Chem,2010,285(19):14071-14077
29. Hennessy BT, Smith DL, Ram PT, et al. Exploiting the PI3K/AKT pathway for cancer drug discovery. Nat Rev Drug Discov 2005; 4:988-1004.
30. Karni R, de Stanchina E, Lowe SW, et al. The gene encoding the splicing factor SF2/ASF is a proto-oncogene. Nat Struct Mol Biol 2007; 14:185-93.
31. Kumar R, Hung MC. Signaling intricacies take center stage in cancer cells. Cancer Res 2005; 65:2511-5.
32. Manning BD, Cantley LC. AKT/PKB signaling: navigating downstream. Cell 2007; 129:1261-74
33.Yim EK, Peng G, Dai H, et al. Rak functions as a tumor suppressor by regulatingPTEN protein stability and function. Cancer Cell 2009; 15:304-14.
34. Marty B,Maire V,Gravier E,et al.Frequent PTEN genomic al-terations and activated phosphatidylinositol 3-kinase pathway in basal-like breast cancer cels[J].Breast Cancer Res,2008,10(6):R101.
35. Stemke-Hale K, Gonzalez-Angulo AM, Lluch A, et al. An integrative genomic and proteomic analysis of PIK3CA, PTEN, and AKT mutations in breast cancer. Cancer Res 2008; 68:6084-91
36. Barbareschi M, Buttitta F, Felicioni L, et al. Different prognostic roles of muta-tions in the helical and kinase domains of the PIK3CA gene in breast carcinomas.Clin Cancer Res 2007; 13:6064-9.
37. Aleskandarany MA,Rakha EA,Ahmed MA, et al.PIK3CA ex-presion in invasive breast cancer: a biomarker of poor prognosis [J].Breast Cancer Res Trea,2009,122(1):45-53.
38. Bachman KE, Argani P, Samuels Y, et al. The PIK3CA gene is mutated with high frequency in human breast cancers. Cancer Biol Ther 2004; 3:772-775.
39. Kirkegard T,Witon CJ,McGlynn LM,et al.AKT activation predicts outcome in breast cancer patients treated with tamoxifen[J].J Path,2005,207(2):139-146.
40. Vasudevan KM,Barbie DA,Davies MA,et al.AKT-independent ignaling downstream of oncogenic PIK3CA mutations in humancancer[J].Cancer Cel,2009,16(1):21-32.
41. Semke-Hale K.An integrative genomic andproteomic analysis of PIK3 CA,PTEN,and AKT mutations inbreast cancer [J].Cancer Res,2008,68(15): 6084-6091
42. Stal O, Perez-Tenorio G, Akerberg L, et al. Akt kinases in breast cancer and the results of adjuvant therapy. Breast Cancer Res 2003; 5:R37-44.
43. Liu H, Radisky DC, Nelson CM, et al. Mechanism of Aktl inhibition of breast cancer cell invasion reveals a protumorigenic role for TSC2. Proc Natl Acad Sci USA 2006; 103:4134-9.
44. Hutchinson JN, Jin J, Cardiff RD, et al. Activation of Akt-1 (PKB-alpha) can ac-celerate ErbB-2-mediated mammary tumorigenesis but suppresses tumor invasion. Cancer Res 2004; 64:3171-8.
45. Maroulakou IG, Oemler W, Naber SP, et al. Aktl ablation inhibits, whereas Akt2 ablation accelerates, the development of mammary adenocarcinomas in mouse mammary tumor virus (MMTV)-ErbB2/neu and MMTV-polyoma middle T transgenic mice. Cancer Res 2007; 67:167-77
46. Berns K, Horlings HM, Hennessy BT, et al. A functional genetic approach identi-fies the PI3K pathway as a major determinant of trastuzumab resistance in breast cancer. Cancer Cell 2007; 12:395-402.
47. Couch FJ, Wang XY, Wu GJ, et al. Localization of PS6K to chromosomal region 17q23 and determination of its amplification in breast cancer. Cancer Res 1999; 59:1408-1411.
48. Barlund M, Forozan F, Kononen J, et al. Detecting activation of ribosomal protein S6 kinase by complementary DNA and tissue microarray analysis. J Natl Cancer Inst 2000; 92:1252-1259.
49. Barlund M, Monni O, Kononen J, et al. Multiple genes at 17q23 undergo amplification and overexpression in breast cancer. Cancer Res 2000; 60: 5340-5344
50. Van der Hage JA, van den Broek LJ, Legrand C, et al. Overexpression of P70 S6 kinase protein is associated with increased risk of locoregional recurrence in node-negative premenopausal early breast cancer patients. Br J Cancer 2004; 90: 1543-1550
51. Walker EH, Pacold ME, Perisic O, et al. Structural determinants of phosphoinositide 3-kinase inhibition by wortmannin, LY294002, quercetin, myricetin, and staurosporine. Mol Cell 2000; 6:909-919
52. Mallucci L, Wells V, Danikas A, Davies D. Turning cell cycle controller genes into cancer drugs. A role for an antiproliferative cytokine (betaGBP). Biochem Pharmacol 2003; 66:1563-1569.
53.Markman B, Serra V, Scaltriti M, et al. Pharmacodynamic (PD) endpoints and cellular proliferation effects of NVP-BEZ235, a dual PI3K/mTOR inhibitor, in breast cancer cells and xenografts with wild-type (WT) and activating PI3K mutations. ASCO Annual Meeting. Clin Oncol 2008; 26:14508.
54. Kondapaka SB, Singh SS, Dasmahapatra GP, Sausville EA, Roy KK. Perifosine, a novel alkylphospholipid, inhibits protein kinase B activation. Mol Cancer Ther 2003; 2:1093-1103.
55. Hennessy BT, Lu Y, Poradosu E, et al. Pharmacodynamic markers of perifosine efficacy. Clin Cancer Res 2007; 13:7421-7431.
56. Castillo SS, Brognard J, Petukhov PA, et al. Preferential inhibition of Akt and killing of Akt-dependent cancer cells by rationally designed phosphatidylinositol ether lipid analogues. Cancer Res 2004; 64:2782-2792.
57. Yang L, Dan HC, Sun M, et al. Akt/protein kinase B signaling inhibitor-2, a selective small molecule inhibitor of Akt signaling with antitumor activity in cancer cells overexpressing Akt. Cancer Res 2004; 64:4394-4399.
58. Wiederrecht GJ, Sabers CJ, Brunn GJ, et al. Mechanism of action of rapamycin:New insights into the regulation of G1-phase progression in eukaryotic cells. Prog Cell Cycle Res 1995; 1:53-71.
59. Thomas GV, Tran C, Mellinghoff IK, et al. Hypoxia-inducible factor determines sensitivity to inhibitors of mTOR in kidney cancer. Nat Med 2006; 12:122-7.
60. Phung TL, Ziv K, Dabydeen D, et al. Pathological angiogenesis is induced by sustained Akt signaling and inhibited by rapamycin. Cancer Cell 2006; 10:159-7
61. Sarbassov DD, Ali SM, Sengupta S, et al. Prolonged rapamycin treatment inhibits mTORC2 assembly and Akt/PKB. Mol Cell 2006; 22:159-68.
62. Stoeltzing O, Meric-Bernstam F, Ellis LM. Intracellular signaling in tumor and endothelial cells:The expected and, yet again, the unexpected. Cancer Cell 2006; 10:89-91
63.Mondesire WH, Jian W, Zhang H, et al. Targeting mammalian target of rapamycin synergistically enhances chemotherapy-induced cytotoxicity in breast cancer cells. Clin Cancer Res 2004; 10:7031-42
64. Johnston SR. Enhancing the efficacy of hormonal agents with selected targeted agents. Clin Breast Cancer 2009; 9(suppl 1):S28-36.
65. Johnston SR. Enhancing the efficacy of hormonal agents with selected targeted agents. Clin Breast Cancer 2009; 9(suppl 1):S28-36.
66. Perez-Tenorio G, Stal O. Activation of AKT/PKB in breast cancer predicts a worse outcome among endocrine treated patients. Br J Cancer 2002; 86:540-5
67. Boulay A, Zumstein-Mecker S, Stephan C, et al. Antitumor efficacy of intermit-tent treatment schedules with the rapamycin derivative RAD001 correlates with prolonged inactivation of ribosomal protein S6 kinase 1 in peripheral blood mononuclear cells. Cancer Res 2004; 64:252-61.
68. Beeram M, Tan QT, Tekmal RR, et al. Akt-induced endocrine therapy resistance is reversed by inhibition of mTOR signaling. Ann Oncol 2007; 18:1323-8
69. Beesisteram M, Tan QT, Tekmal RR, et al. Akt-induced endocrine therapy rance is reversed by inhibition of mTOR signaling. Ann Oncol 2007; 18:1323-8
70. Baselga J, Semiglazov V, van Dam P, et al. Phase Ⅱ randomized study of neoad-juvant everolimus plus letrozole compared with placebo plus letrozole in patients with estrogen receptor-positive breast cancer. J Clin Oncol 2009; 27:2630-7。
71. Johnston SR. New strategies in estrogen receptor-positive breast cancer. Clin Cancer Res 2010; 16:1979-87
72. Loi S, Haibe-Kains B, Majjaj S, et al. PIK3CA mutations associated with gene signature of low mTORC1 signaling and better outcomes in estrogen receptor-positive breast cancer. Proc Natl Acad Sci U S A 2010; 107:10208-13
73. Shi Y, Yan H, Frost P, et al. Mammalian target of rapamycin inhibitors activate the AKT kinase in multiple myeloma cells by up-regulating the insulin-like growth factor receptor/insulin receptor substrate-1/phosphatidylinositol 3-kinase cascade. Mol Cancer Ther 2005; 4:1533-40
74. Wan X, Harkavy B, Shen N, et al. Rapamycin induces feedback activation of Akt signaling through an IGF-1R-dependent mechanism. Oncogene 2007; 26:1932-40.
75. Meric-Bernstam F, Gonzalez-Angulo AM. Targeting the mTOR signaling network for cancer therapy. J Clin Oncol 2009; 27:2278-87.
76. Hoeflich KP, O'Brien C, Boyd Z, et al. In vivo antitumor activity of MEK andphosphatidylinositol 3-kinase inhibitors in basal-like breast cancer models. Clin Cancer Res 2009; 15:4649-64.
77. Saal LH, Gruvberger-Saal SK, Persson C, et al. Recurrent gross mutations of thePTEN tumor suppressor gene in breast cancers with deficient DSB repair. NatGenet 2008; 40:102-7.79.
78.Mirzoeva OK, Das D, Heiser LM, et al. Basal subtype and MAPK/ERK kinase(MEK)-phosphoinositide 3-kinase feedback signaling determine susceptibility ofbreast cancer cells to MEK inhibition. Cancer Res 2009; 69:565-72
79. Wang X, Sun SY, et al. Enhancing mTOR-targeted cancer therapy. Expert Opinion on Therapeutic Targets, 2009,13(10):1193-1203,2009.
80. Lopiccolo J, Blumenthal GM, Bernstein WB, et al. Targeting the PI3K/Akt/mTOR pathway: effective combinations and clinical considerations. Drug Resistance Updates, 2008,11(1-2):32-50
81. Chiang GG, Abraham RT. Targeting the mTOR signaling network in cancer," Trends in Molecular Medicine, 2007,13(10):433-442,.
82. Abraham RT, Gibbons JJ. The mammalian target of rapamycin signaling pathway: twists and turns in the road to cancer therapy. Clinical Cancer Research, 2007, 13(11):3109-3114.
83. Choo AH, Blenis, J. Not all substrates are treated equally Implications for mTOR, rapamycin-resistance and cancer therapy," Cell Cycle, 2009,8(4): 567-572.
84. Schalm SS, J. Blenis. Identification of a conserved motif required for mTOR signaling. Current Biology, 2002,12(8):632-639.
85. Schalm S, Fingar, DF. Sabatini M, et al. TOS motif-mediated raptor binding regulates 4E-BP1 multisite phosphorylation and function. Current Biology, 2003,13(10):797-806
86. Wang X, Yue SY, Fu H, et al. Enhancing mammalian target of rapamycin (mTOR)-targeted cancer therapy by preventing mTOR/raptor inhibition-initiated, mTOR/rictor-independent Akt activation," Cancer Research, 2008, 68(18):7409-7418,
87. Wang X, Sun YH, Enhancing mTOR-targeted cancer therapy. Expert Opinion on Therapeutic Targets, 2008,13(10):1193-1203.
88. Jansen AP, Camalier CE, Stark C,et al. Characterization of programmed cell death 4 in multiple human cancers reveals a novel enhancer of drug sensitivity. Mol Cancer Ther, 2004,103:177-185
89. Afonia O, Juste D, Dan S, et al. Induction of PDCD4 tumor suppressor gene expression by RAR agonists, antiestrogen and HER-2/neu antagonist in breast cancer cells. Evidence for a role in apoptosis. Oncogene, 2004,23:8135-8157
90. Palamarchuk A, Efanov A, Maximov V, et al. Akt Phosphorylates and Regulates Pdcd4 Tumor Suppressor Protein[J].CancerResearch,2005,65(24):11282-11286.
91.Woodard J, Sassano A, Hay N, et al. Statin-dependent suppression of the Akt/mammalian target of rapamycin signaling cascade and programmed cell death 4 up-regulation in renal cell carcinoma[J]. Clin Cancer Res,2008,14(14):4640-4649.
1.朱冬兵,陈莉.乳腺癌C-erbB-2、nm23基因表达与免疫活性细胞反应的相关性[J].中国肿瘤,2007,16(6)：439441.
2. Grey SR,Dlay SS,Leone BE,et al.Prediction of nodal spread of breast cancer by using artificial neural network-based analyses of S100A4,nm23 and steroid receptor expression.Clin ExpMetastasis,2003,20(6):507-514.
3. HabashyHO, Powe DG, Rakha EA, et a.l Forkhead-box A1(FOXA1) expression in breast cancer and its prognostic signifi-cance[J].Eur JCancer, 2008,44(11): 1541-1551.
4. Wolf I, Bose S,W illiamson EA, et a.l FOXA1:growth inhibitor and a favorable prognostic factor in human breast cancer[J]. Int J Cancer, 2007,120(5): 1013-1022.
5. Sorlie T.Molecular portraits of breast cancer: tumour subtypes asdistinctdisease entities[J]. Eur J Cancer, 2004,40(18):2667-2675
6. Sotiriou C,W irapati P, Loi S, et a.l Gene expression profiling inbreast cancer: understanding themolecular basis ofhistologic gradeto improve prognosis[J]. JNatlCancer Inst,2006,98(4):262-272.
7. Jakowlew SB. Transforming growth factor-beta incancer and metastasis[J].Cancer Metastasis Rev, 2006,25(3):435-457.
8. Galliher AJ, Neil JR, Schiemann WP. Role of transforming growth factor-beta in cancer progression[J]. Future Onco,l 2006,2(6):743-763
9. Marchetti P, Cannita K,Ricevuto E,et al. Prognostic value of p53 molecular status in high-risk primary breast cancer.Ann Oncol,2003,14(5):704-708.
10. Poelman SM, Heimann R, Fleming GF, et al. Invariant p53 immunostaining in primary and recurrent breast cancer.Eur J Cancer,2004,40(l):28-32.
11.Linjawi A, Kontogiannea M, Halwani F, et al. Prognostic significance of p53,bcl-2,and Bax expression in early breast cancer. J Am Coll Surg, 2004,198(1):83-90.
12. Shibahara K, Asano M, Ishida Y, et al. Isolation of a novel mouse gene MA-3 that is induced upon programmed cell death. Gene, 1995,166(2):297-301
13. Onishi Y, Hashimoto S, Kizaki H, et al. Cloning of the TIS gene suppressed by topoisomerase inhibitors. Gene, 1998,215(2):453-459
14. Azzoni L, Zatsepina O, Abede B, et al. Differential transcriptional regulation of CD161 and a novel gene, 197/15a, by IL-2,IL-15, and IL-12 in NK and T cells. J Immunal,1998; 161;3493-3498
15. Zhang Z, Kubois RN. Detection of differentially expressed genes in human colon carcinoma cells treated with a selective COX-2 inhibition Oncogene,2001, 20:44504458
16. Schlichter U, Burk O, Worpenberg C, et al. The chicken PDCD4 gene is regulated by v-Myb. Oncogene, 2001,20:231-236
17. Cmarik J L, Min H Z, Hegamyer G, et al. Differentially expressed protein Pdcd4 inhibits tumor prmoter-induced neoplastic transformation. Proc Natl Acad Sci USA,1999,96 (24):14037-14042
18. Jansen AP, Camalier CE, Stark C,et al. Characterization of programmed cell death 4 in multiple human cancers reveals a novel enhancer of drug sensitivity. Mol Cancer Ther, 2004,103:
19. Halliard A, Halliard B, Zheng SJ, et al. Translational regulation of autoimmune inflammation and lymphoma genesis. J Immunol,2006,177:8095-902
20. Gao F, Wang X, Zhu F, et al. PDCD4 gene silencing in gliomas is associated with 5'CpG island methylation and unfavourable prognosis[J]. Journal of Cellular and Molecular Medicine,2009,13(10):4257-4267.
21. Chen Y, Knosel T, Kristiansen G, et al. Loss of PDCD4 expression in human lung cancer correlates with tumour progression and prognosis[J]. J Pathol,2003,200:640-646.
22. Li X, Xin S, Yang D, et al. Down-regulation of PDCD4 expression is an independent predictor of poor prognosis in human renal cell carcinoma patients[J]. J Cancer Res Clin Oncol,2012,138(3):529-535.
23. Wen Y H, Shi X, Chiriboga L, et al. Alterations in the expression of PDCD4 in ductal carcinoma of the breast[J]. Oncol Rep,2007,18(6):1387-1393.
24. Asangani I A, Rasheed S A, Nikolova D A, et al. MicroRNA-21 (miR-21) post-transcriptionally downregulates tumor suppressor Pdcd4 and stimulates invasion, intravasation and metastasis in colorectal cancer[J]. Oncogene,2008,27:2128-2136.
25. Motoyama K, Inoue H, Mimori K, et al. Clinicopathological and prognostic significance of PDCD4 and microRNA-21 in human gastric cancer[J]. Int J Oncol,2010,36:1089-1095.
26. Ma G, Guo K J, Zhang H, et al. [Expression of programmed cell death 4 and its clinicopathological significance in human pancreatic cancer][J]. Zhongguo Yi Xue Ke Xue Yuan Xue Bao,2005,27(5):597-600.
27. Zhang H, Ozaki I, Mizuta T, et al. Involvement of programmed cell death 4 in transforming growth factor-betal-induced apoptosis in human hepatocellular carcinoma[J]. Oncogene,2006,25(45):6101-6112.
28. Reis P, Tomenson M, Cervigne N, et al. Programmed cell death 4 loss increases tumor cell invasion and is regulated by miR-21 in oral squamous cell carcinoma[J]. Molecular Cancer,2010,9(1):238-243.
29. Wei N A, Liu S S, Leung T H, et al. Loss of Programmed cell death 4 (Pdcd4) associates with the progression of ovarian cancer[J]. Mol Cancer,2009,8:70-79.
30. Afonia O, Juste D, Dan S, et al. Induction of PDCD4 tumor suppressor gene expression by RAR agonists, antiestrogen and HER-2/neu antagonist in breast cancer cells. Evidence for a role in apoptosis. Oncogene, 2004,23:8135-8157
31. Wen Y H, Shi X, Chiriboga L, et al. Alterations in the expression of PDCD4 in ductal carcinoma of the breast[J]. Oncol Rep,2007,18(6):1387-1393
32. Chen Y, Knosel T, Kristiansen G, et al. Loss of PDCD4 expression in human lung cancer dorrelates with tumour progression and prognosis. J Pathol,2003, 200:640-655
33. Ma G, Guo K J, Zhang H, et al. [Expression of programmed cell death 4 and its clinicopathological significance in human pancreatic cancer][J]. Zhongguo Yi Xue Ke Xue Yuan Xue Bao,2005,27(5):597-600.
34. Yang H S, Jansen A P, Komar A A, et al. The transformation suppressor Pdcd4 is a novel eukaryotic translation initiation factor 4A binding protein that inhibits translation[J]. Mol Cell Biol,2003,23(1):26-37.
35. Yang H S, Matthews C P, Clair T, et al. Tumorigenesis suppressor Pdcd4 down-regulates mitogen-activated protein kinase kinase kinase kinase 1 expression to suppress colon carcinoma cell invasion[J]. Mol Cell Biol,2006, 26:1297-1306
36. Leupold J H, Yang H S, Colburn N H, et al. Tumor suppressor Pdcd4 inhibits invasion/intravasation and regulates urokinase receptor (u-PAR) gene expression via Sp-transcription factors[J]. Oncogene,2007,26:4550-4562
37. Yang HS, Knies JL, Stark C, et al. PDCD4suppresses tumor phenotype in JB6 cells by inhibiting AP-1 transactivation. Oncogene,2003,22:3712-3726
38. Nieves-Alicea R, Colburn N H, Simeone A M, et al. Programmed cell death 4 inhibits breast cancer cell invasion by increasing tissue inhibitor of metalloproteinases-2 expression[J]. Breast Cancer Res Treat,2009,114(2): 203-209.
39. Bitomsky N, Wethkamp N, Marikkannu R, et al. siRNA-mediated knockdown of Pdcd4 expression causes upregulation of p21(Wafl/Cipl) expression[J]. Oncogene,2008,27(35):4820-4829
40. Jin H, Kim T H, Hwang S K, et al. Aerosol delivery of urocanic acid-modified chitosan/programmed cell death 4 complex regulated apoptosis, cell cycle, and angiogenesis in lungs of K-ras null mice[J]. Mol Cancer Ther,2006,5:1041-1049.
41. Effects of chemotherapy and hormonal therapy for early breast cancer on recurrence and 15-year survival:an overview of the randomised trials[J]. Lancet,2005,365(9472):1687-1717.
42. Asangani I A, Rasheed S A, Nikolova D A, et al. MicroRNA-21 (miR-21) post-transcriptionally downregulates tumor suppressor Pdcd4 and stimulates invasion, intravasation and metastasis in colorectal cancer[J]. Oncogene, 2008,27:2128-2136
43. Chen Y, Liu W, Chao T, et al. MicroRNA-21 down-regulates the expression of tumor suppressor PDCD4 in human glioblastoma cell T98G[J]. Cancer Lett,2008,272(2):197-205.
44. Reis P, Tomenson M, Cervigne N, et al. Programmed cell death 4 loss increases tumor cell invasion and is regulated by miR-21 in oral squamous cell carcinoma[J]. Molecular Cancer,2010,9(1):238.
45. Wang J, Li Y, Wang X, et al. Ursolic Acid Inhibits Proliferation and Induces Apoptosis in Human Glioblastoma Cell Lines U251 by Suppressing TGF-β1/miR-21/PDCD4 Pathway[J]. Basic & Clinical Pharmacology & Toxicology,2012,111(2):106-112.
46. Zhang H, Ozaki I, Mizuta T, et al. Involvement of programmed cell death 4 in transforming growth factor-betal-induced apoptosis in human hepatocellular carcinoma[J]. Oncogene,2006,25(45):6101-6112
47. Dorrello N V, Peschiaroli A, Guardavaccaro D, et al. S6K1- and betaTRCP-mediated degradation of PDCD4 promotes protein translation and cell growth[J]. Science,2006,314(5798):467-471.
48. Liwak U, Thakor N, Jordan L E, et al. Tumor suppressor PDCD4 represses internal ribosome entry site-mediated translation of antiapoptotic proteins and is regulated by S6 kinase 2[J]. Mol Cell Biol,2012,32(10):1818-1829.
49. Palamarchuk A, Efanov A, Maximov V, et al. Akt Phosphorylates and Regulates Pdcd4 Tumor Suppressor Protein[J]. Cancer Research,2005,65(24):11282-11286.
50. Woodard J, Sassano A, Hay N, et al. Statin-dependent suppression of the Akt/mammalian target of rapamycin signaling cascade and programmed cell death 4 up-regulation in renal cell carcinoma[J]. Clin Cancer Res,2008, 14(14):4640-4649.
51.Carayol N, Katsoulidis E, Sassano A, et al. Suppression of Programmed Cell Death 4 (PDCD4) Protein Expression by BCR-ABL-regulated Engagement of the mTOR/p70 S6 Kinase Pathway [J]. Journal of Biological Chemistry,2008, 283(13):8601-8610.
52. Schmid T, Jansen A P, Baker A R, et al. Translation inhibitor Pdcd4 is targeted for degradation during tumor promotion[J]. Cancer Res,2008,68:1254-1260
53. Grant S. Cotargeting survival signaling pathways in cancer[J]. J Clin Invest,2008,118(9):3003-3006.
54. Zhang Z, Dubois R N. Detection of differentially expressed genes in human colon carcinoma cells treated with a selective COX-2 inhibitor[J]. Oncogene,2001, 20(33):4450-4456.
55. Schlichter U, Burk O, Worpenberg S, et al. The chicken Pdcd4 gene is regulated by v-Myb[J]. Oncogene,2001,20(2):231-239.
56.马刚,张浩,董明,等。大肠癌组织中PDCD4表达的临床病理学意义及其对预后的影响。中国现代医学杂志,2007,17(11)：1281-1286
57. CHen Y, Knosel T, Kristiansen G, et al. Loss of Pdcd4 expression in human lung cancer correlates with tumor progression and prognosis[J]. J Pathol, 2003,200(5):640-646.
58. Wei ZT, Zhang X, Wang XY, et al. Pdcd4 inhibits the malignant phenotype of ovarian cancer cells [J]. Cancer Sci, 2009,100(8):1408-1413
59. Zhang Z, DuBois RN. Detection of differentially expressed genes in human colon carcinoma cells treated with a selective COX-2 inhibitor. Oncogene 2001;20(33):4450-4456.
60. Leupold JH, Yang HS, Colburn NH, et al. Tumor suppressor Pdcd4 inhibits invasion/intravasation and regulates urokinase receptor (u-PAR) gene expression via Sp-transcription factors. Oncogene 2007;26(31):4550-4562.
61. Yang HS, Matthews CP, Clair T, et al. Tumorigenesis suppressor Pdcd4 down-regulates mitogen-activated protein kinase kinase kinase kinase 1 expression to suppress colon carcinoma cell invasion. Mol Cell Biol 2006;26(4):1297-1306.
62. Wang Q, Sun Z, Yang HS. Downregulation of tumor suppressor Pdcd4 promotes invasion and activates both beta-catenin/Tcf and AP-1-dependent transcription in colon carcinoma cells. Oncogene 2008;27(11):1527-1535.
63. Wen YH, Shi X, Chiriboga L, et al. Alterations in the expression of PDCD4 in ductal carcinoma of the breast. Oncol Rep 2007; 18(6):1387-1393
64. Gonzalez VV, Nieves AR, McMurty V, et al. Programmed cell death 4 inhibits leptin-induced breast cancer cell invasion. Oncol Rep,2012,27(3):861-866
65. Rene NA, Nancy HC, Ann MS, et al. Programmed cell death 4 inhibits breast cancer cell invasion by increasing Tissue inhibitor of Metalloproteinases-2 expression. Breast can Res,2009;114(2):203-209。
66. Palamarchuk K, Efanov A, MAXIMOV V, et al. Akt phosphorylates and regulates Pdcd4 tumor suppressor protein[J]. Cancer Re, 2005,65(24):11282-11286.
67.许传兵,徐忠华,刘照旭等。抑癌基因Pdcd4在肾透明细胞癌中的表达及生物学意义。中国现代医学杂志,2010,20(18)：2775-2778
68. Frankel LB, Christoffersen NR, Jacobsen A, et al. Programmed Cell Death 4 (PDCD4) is an important functional target of the MicroRNA miR-21 in breast cancer cells. J Biol Chem 2008;283(2):1026-1033.
1.Shibahara K, Asano M, Ishida Y, et al. Isolation of a novel mouse gene MA-3 that is induced upon programmed cell death. Gene, 1995,166(2):297-301
2.Chen Y, Knosel T, Kristiansen G, et al. Loss of PDCD4 expression in human lung cancer correlates with tumour progression and prognosis[J]. J Pathol,2003,200:640-646.
3. Wei N A, Liu S S, Leung T H, et al. Loss of Programmed cell death 4 (Pdcd4) associates with the progression of ovarian cancer[J]. Mol Cancer,2009,8:70-79
4. Zhang Z, Kubois RN. Detection of differentially expressed genes in human colon carcinoma cells treated with a selective COX-2 inhibition Oncogene, 2001,20:44504458
5.Leupold JH, Yang HS, Colburn NH, et al. Tumor suppressor Pdcd4 inhibits invasion/intravasation and regulates urokinase receptor (u-PAR) gene expression via Sp-transcription factors. Oncogene 2007;26(31):4550-4562.
6.Yang HS, Matthews CP, Clair T, et al. Tumorigenesis suppressor Pdcd4 down-regulates mitogen-activated protein kinase kinase kinase kinase 1 expression to suppress colon carcinoma cell invasion. Mol Cell Biol 2006;26(4):1297-1306.
7. Wang Q, Sun Z, Yang HS. Downregulation of tumor suppressor Pdcd4 promotes invasion and activates both beta-catenin/Tcf and AP-1-dependent transcription in colon carcinoma cells. Oncogene 2008;27(11):1527-1535.
8. Wen Y H, Shi X, Chiriboga L, et al. Alterations in the expression of PDCD4 in ductal carcinoma of the breast[J]. Oncol Rep,2007,18(6):1387-1393.
9.Nieves-Alicea R, Colburn N H, Simeone A M, et al. Programmed cell death 4 inhibits breast cancer cell invasion by increasing tissue inhibitor of metalloproteinases-2 expression[J]. Breast Cancer Res Treat,2009,114(2): 203-209.
10. Jansen AP, Camalier CE, Stark C,et al. Characterization of programmed cell death 4 in multiple human cancers reveals a novel enhancer of drug sensitivity. Mol Cancer Ther,2004,103-109
11. Afonia O, Juste D, Dan S, et al. Induction of PDCD4 tumor suppressor gene expression by RAR agonists, antiestrogen and HER-2/neu antagonist in breast cancer cells. Evidence for a role in apoptosis. Oncogene, 2004,23:8135-8157
12. Palamarchuk A, Efanov A, Maximov V, et al. Akt Phosphorylates and Regulates Pdcd4 Tumor Suppressor Protein[J].CancerResearch,2005,65(24):11282-11286
13.Woodard J, Sassano A, Hay N, et al. Statin-dependent suppression of the Akt/mammalian target of rapamycin signaling cascade and programmed cell death 4 up-regulation in renal cell carcinoma[J]. Clin Cancer Res,2008,14(14): 4640-4649
14. Foulkes WD, Smith IE, Reis-Filho JS. Triple-negative breast cancer [J]. N Engl J Med,2010,363(20):1938-1948.
15.O'Shaughnessy J, Osborne C, Pippen JE, et al. Iniparib plus chemotherapy in metastatic triple-negative breast cancer[J]. N Engl J Med,2011,364(3):205-214.
16. Metzger-Filho O, Tutt A, de Azambuja E, et al. Dissecting the heterogeneity of triple-negative breast cancer[J]. J Clin Oncol,2012,30(15):1879-1887.
17. Millikan RC, Newman B, Tse CK, et al. Epidemiology of basal-like breast cancer[J]. Breast Cancer Res Treat,2008,109:123-139.
18. Dent R, Hanna WM,Trudeau M, et al. Pattern of metastatic spread in triple-negative breast cancer [J]. Breast Cancer ResTreat,2009,115(2):423-428
19. Bauer KR, Brown M, Cress RD, et al. Descriptive analysis of estrogen receptor(ER)-negative, progesterone receptor(PR)-negative, and HER2-negative invasive breast cancer, the so-called triple-negative phenotype:a population-based study from the California Cancer Registry[J]. Cancer,2007, 109(9):1721-1728.
20. Kim MJ, Ro JY, Ahn SH, et al. Clinicopathologic significane of the basal-like subtype of breast cancer:A conparison with hormone receptor and Her2/ neuover expressing phenotypes[J]. Hum Pathol,2006,37(9):1217-1226
21. Sara M, Bando Y, Takahashi M, et al. Screening for basal marker expression is necessary for decision of the rapeutic strategy for triple-negative breast cancer [J]. J Surg Oncol,2008,97(1):30-34.
22. LiuZB, Liu G Y, Yang W R, et al. Triple-negative breast cancer types exhibit a distinct poor clinical characteristic in lymph node-negative Chinese patients[J]. Oncol Rep,2008,20(4):987-994.
23. Rakha EA, El-Sayed ME, Green AR, et al. Prognostic markers in triple-negative breast cancer[J]. Cancer,2007,109(1):25-32
24. Musgrove EA, Sutherland RL. Biological determinants of endocrine resistance in breast cancer. Nat Rev Cancer 2009;9:631-43
25. Cui X, Zhang P, Deng W, et al. Insulin-like growth factor-I inhibits progesterone receptor expression in breast cancer cells via the phosphatidylinositol 3-kinase/Akt/mammalian target of rapamycin pathway:progesterone receptor as a potential indicator of growth factor activity in breast cancer. Mol Endocrinol 2003; 17:575-88.
26. Ellis MJ, Ding L, Shen D, Luo J, Suman VJ, et al. Whole-genome analysis informs breast cancer response to aromatase inhibition. Nature,2012,486: 3532360
27. Stephens PJ, Tarpey PS, Davies H, Van Loo P, Greenman C, et al. The landscape of cancer genes and mutational processes in breast cancer. Nature,2012,486:4002404.
28. TCGA. Comprehensive molecular portraits of human breast tumours. Nature,2012, 490:61270.
29.Kalinsky K, Heguy A, Bhanot UK, Patil S, Moynahan ME (2011) PIK3CA mutations rarely demonstrate genotypic intratumoral heterogeneity and are selected for in breast cancer progression. Breast Cancer Res Treat 129:6352643
30. Crowder RJ, Phommaly C, Tao Y, et al. PIK3CA and PIK3CB inhibition produce synthetic lethality when combined with estrogen deprivation in estrogen receptor-positive breast cancer. Cancer Res,2009,69:3955-3962.
31. Yu K, Toral-Barza L, Discafani C, et al. mTOR, a novel target in breast cancer: the effect of CCI-779, an mTOR inhibitor, in preclinical models of breast cancer. Endocr Relat Cancer 2001; 8:249.
32. Simoncini T, Hafezi-Moghadam A, Brazil DP, et al. Interaction of oestrogen receptor with the regulatory subunit of phosphatidylinositol-3-OH kinase. Nature 2000;407:538.
33. Clemens, M. J., and Bommer, U. A. Translational control:the cancer connection. Int. J. Biochem. Cell Biol.,31:1-23,1999.
34. Dufner, A., Andjelkovic, M., Burgering, B. M., Hemmings, B. A., and Thomas, G. Protein kinase B localization and activation differen-tially affect S6 kinase 1 activity and eukaryotic translation initiation factor 4E-binding protein 1 phosphorylation. Mol. Cell. Biol.,19:4525-4534,1999
35. Peterson, R. T., Desai, B. N., Hardwick, J. S., and Schreiber, S. L. Protein phosphatase 2A interacts with the 70-kDa S6 kinase and is activated by inhibition of FKBP12-rapamycin-associated protein. Proc. Natl. Acad. Sci. USA, 96:4438-4442,1999
36. Jefferies, H. B., Fumagalli, S., Dennis, P. B., Reinhard, C., Pearson, R. B., and Thomas, G. Rapamycin suppresses 5□TOP mRNA translation through inhibition of p70s6k. EMBO J.,16:3693-3704,1997
37. Sekulic, A., Hudson, C. C., Homme, J. L., Yin, P., Otterness, D. M., Karnitz, L. M., et al. A direct linkage between the phosphoinositide 3-kinase-AKT signaling pathway and the mammalian target of rapamy-cin in mitogen-stimulated and transformed cells. Cancer Res., 60:3504-3513,2000
38. Reynolds, T. H. T., Bodine, S. C., and Lawrence, J. C., Jr. Control of Ser2448 phosphorylation in the mammalian target of rapamycin by insulin and skeletal muscle load. J. Biol. Chem.,277:17657-17662,2002.
39. Inoki, K., Li, Y., Zhu, T., Wu, J., and Guan, K. L. TSC2 is phosphorylated and inhibited by Akt and suppresses mTOR signalling. Nat. Cell Biol.,4:648-657, 2002.
40. Weng, Q. P., Kozlowski, M., Belham, C., Zhang, A., Comb, M. J., and Avruch, J. Regulation of the p70 S6 kinase by phosphorylation in vivo. Analysis using site-specific anti-phosphopeptide antibodies. J. Biol. Chem., 273:16621-16629, 1998
41. Sarbassov DD, Ali SM, Kim DH et al. Rictor, a novel binding partner of mTOR, defines a rapamycin-insensitive and raptor-independent pathway that regulates the cytoskeleton. Curr Biol 2004;14:1296-1302.
42. Huang J, Dibble CC, Matsuzaki M et al. The TSC1-TSC2 complex is re-quired for proper activation of mTOR complex 2. Mol Cell Biol 2008;28:4104-4115.
43. Sarbassov DD, Guertin DA, Ali SM et al. Phosphorylation and regulation of Akt/PKB by the rictor-mTOR complex. Science 2005;307:1098-1101
44. Walsh S, Flanagan L, Quinn C, et al. mTOR in breast cancer: differential expression in triple-negative and non-triple-negative tumors. Breast 2012, 21(2):178-182.
45. Shir-Hwa U, Shin-Cheh C, Yu-Sun Chang, et al. Phosphorylated mTOR expression correlates with poor outcome in early-stage triple negative breast carcinomasInt J Clin Exp Pathol 2012;5(8):806-813
46. Kuo CJ, Chung J, Fiorentino DF et al. Rapamycin selectively inhibits in-terleukin-2 activation of p70 S6 kinase. Nature 1992;358:70-73
47. Meric-Bernstam F, Gonzalez-Angulo AM. Targeting the mTOR signaling network for cancer therapy. J Clin Oncol 2009;27:2278-2287
48. LearyA,DowsettM.Combinationtherapywitharomataseinhibitors:the next era of breast cancer treatment? Br J Cancer 2006;95:661-666
49. SunSY,RosenbergLM,WangXetal.ActivationofAktandeIF4Esur-vival pathways by rapamycin-mediated mammalian target of rapamycin inhibition. Cancer Res 2005;65:7052-7058.
50. Lindsley CW. The Akt/PKB family of protein kinases:a review of small molecule inhibitors and progress towards target validation:a 2009 update. Curr Top Med Chem 2010;10:458-477
51,Ozbudak IH, Karaveli S, Simsek T,et al. Neoangiogenesisand expression of hypoxia-inducible factorlalpha, vascular endothelial growth factor, and glucose transporter-lin endom-etrioid type endometrium adenocarcinomas[J]. Gynecol Oncol,2008,108(3):603-608.
52. Li W, Petrimpol M, Molle KD,et al. Hypoxia-induced endothelial proliferation requires both mTORCland mTORC2[J].Circ Res,2007,100(1):79-872
53. Hashemolhosseini S, NagamineY, Morley SJ, et a.l Rapamycininhibition of theGl toS transition ismediated by effectson cyclinDl mRNA and protein stability [J]. J Biol Chem, 1998,273
54. Proud CG, Denton RM. Molecularmechanisms for the control oftranslation by insulin [J]. Biochem J, 1997,328(pt2):329-341
55.Tolcher AW. Novel therapeutic molecular targets for prostatecancer: themTOR signaling pathway and epidermal growth factor receptor [J]. J Uro,l 2004, 171(2pt2):S41-S43.
56. Huang S, Shu L, DillingMB, et a.l Sustained activation of theJNK cascade and rapamycin induced apoptosis are suppressed by p53/p21(Cipl) [J]. MolCel,l 2003,11(6):1491-1501.
57. Yang, H.S., Jansen, A.P., Komar, A.A., Zheng, X., Merrick, W.C., Costes, S., Lockett, S.J., Sonenberg, N. and Colburn, N.H. (2003a) The transformation suppressor Pdcd4 is a novel eukaryotic translation initiation factor 4A binding protein that inhibits translation. Mol. Cell. Biol. 23,26-37
58.]Mudduluru, G., Medved, F., Grobholz, R., Jost, C., Gruber, A., Leupold, J.H., Post, S., Jansen, A., Colburn, N.H. and Allgayer, H. (2007) Loss of programmed cell death 4 expression marks adenoma-carcinoma transition, correlates inversely with phosphorylated protein kinase B, and is an independent prognostic factor in resected colorectal cancer. Cancer 110, 1697-1707
59. Gezeh PT, Elin K, Marie AW,et al. Clinical potential of the mTOR targets S6K1 and S6K2 in breast cancer. Breast Can Res Treat,2011,128:713-723。
60. Sehgal SN. Sirolimus:its discovery, biological properties, and mechanism of action. Transplant Proc 2003;35:7S.
61. Monks A, Scudiero D, Skehan P, et al. Feasibility of a high-flux anticancer drug screen using a diverse panel of cultured human tumor cell lines. J Natl Cancer Inst1991;83:757.
62. Dancey JE. Clinical development of mammalian target of rapa-mycin inhibitors. Hematol Oncol Clin North Am 2002; 16:1101
63. Sharon BPenelope MAlexander MRapamycin Inhibits Proliferation of Estrogen-Receptor-Positive Breast Cancer Cells Journal of Surgical Research 138,37-44 (2007)
64. Yoon SO, Roux PP.Rapamycin resistance:mTORC1 substrates hold some of the answers.Curr Biol.2013 Oct 7;23(19):R880-3.
65.B.Hassan,A.Akcakanat,S.Takafumi,MTORInhibitorMLN0128hasAntitumorEffic acyinCell Lines With Intrinsic and Acquired Rapamycin-Resis-tance。 ASSOCIATION FOR ACADEMIC SURGERY AND SOCIETY OF UNIVERSITY SURGEONS; 2010
2.Guillermet G J, Bjorklof K, Salpekar A, et al. The p110beta isoform of phosphoinositide 3-kinase signals downstream of G protein-coupled receptors and is functionally redundant with pllOgamma. ProcNatl Acad Sci,2008; 105(24):8292-8297
3. Shaw RJ, Cantley LC. Ras, PI(3)K and mTOR signalling controls tumour cell growth. Nature. 2006; 441(7092):424-430.
4.Skolnik EY, Margolis B, Mohammadi M, et al. Cloning of PI3 kinase-associated p85 utilizing a novel method for expression/cloning of target proteins for receptor tyrosine kinases. Cell. 1991;65(1):83-90.
5.Maehama T, Dixon JE. Thetumorsuppressor,PTEN/MMAC1,dephosphorylates the lipid second messenger, phosphatidylinositol3,4,5-trisphosphate. J Biol Chem. 1998;273(22):13375-13378.
6.Cheng JQ, Lindsley CW, Cheng G.Z, et al. The Akt/PKB pathway:molecular target for cancer drug discovery. Oncogene, 2005; 24(50):7482-7492,
7.Sokunaga E,Oki E, Egashira A,et al.Deregulation of the Akt pathway in human cancer[J].Cur Cancer Drug Targets,2008,8(1):27-36
8.Feng J, Park J, Cron P, et al. Identification of a PKB/Akt hydrophobic motif Ser-473 kinase as DNA-dependent protein kinase. J Biol Chem.2004; 279(39):41189-96.
9.Hresko RC, Mueckler M. mTOR.RICTOR is the Ser473 kinase for Akt/protein kinase B in 3T3-L1 adipocytes. J Biol Chem. 2005; 280(49):40406-16.
10. Wullschleger S, Loewith R, Hall MN. TOR signaling in growth and metabolism. Cell 2006; 124:471-84.
11. Wang L, Harris TE, Lawrence JC, Jr. Regulation of proline-rich Akt substrate of 40 kDa (PRAS40) function by mammalian target of rapamycin complex 1 (mTORCl)-mediated phosphorylation. J Biol Chem 2008; 283:15619-27.
12. MericBF, Gonzalez AM. Targeting the mTOR signaling network for cancer therapy. J Clin Oncol 2009; 27:2278-87.
13.Benedetti A, Graff JR. eIF-4E expression and its role in malignancies and metastases. Oncogene 2004; 23:3189-99
14. Jastrzebski K, Hannan KM, Tchoubrieva EB, et al. Coordinate regulation of ribo-some biogenesis and function by the ribosomal protein S6 kinase, a key mediator of mTOR function. Growth Factors 2007; 25:209-26.
15. Nakamura JL, Garcia E, Pieper RO. S6K1 plays a key role in glial transformation. Cancer Res 2008; 68:6516-23
16. Barlund M, Forozan F, Kononen J, et al. Detecting activation of ribosomal protein S6 kinase by complementary DNA and tissue microarray analysis. J Natl Cancer Inst 2000; 92:1252-9
17. Garc A, Martinez JM,Aleesi DR. mTOR complex 2 (mTORC2) controls hydrophobic motif phosphorylation and activation of serum-and glucocorticoid-induced protein kinase 1 (SGK1)[J]. ChemJ,2008,416(3):375-385.
18. Jacinto E, Facchinetti V, Liu D, et al. SIN1/MIP1 maintains rictor-mTOR com-plex integrity and regulates Akt phosphorylation and substrate specificity. Cell 2006; 127:125-37.
19. Sarbassov DD, Ali SM, Kim DH, et al. Rictor, a novel binding partner of mTOR, defines a rapamycin-insensitive and raptor-independent pathway that regulates the cytoskeleton. Curr Biol 2004; 14:1296-302.
20. Hernandez-Negrete I, Carretero-Ortega J, Rosenfeldt H, et al. P-Rexl links mammalian target of rapamycin signaling to Rac activation and cell migration. J Biol Chem 2007; 282:23708-15
21.Noh EM,Le YR,Chay KO,et al.PTEN and the PI3-kinasepathway in cancer[J].Ann Review Pathol,2009,4(2):127-150
22. Demidenko ZN,Shutman M,Blagosklonnyl MV. Pharmacologic inhibition of MEK and PI-3K converges on the mTOR/S6 pathway to decelerate cellular senescence[J]. Cell Cycle, 2009,8(12):1896-1900
23.JACINTO E,LOEWITH R,SCHMIDT A,et al. Mammalian TOR complex 2 controls the actin cytoskeleton and is rapamycin insensitive [J]. Nat Cell Biol,2004,6(11):1122-1128.
24. Sarbassov DD,Ali SM,Kim DH,et al. Rictor,a novel binding partner of mTOR, defines a rapamycin-insensitive and raptor-independent pathway that regulates the cytoskeleton[J]. Curr Biol,2004,14(14):1296-1302
25. Rosner M,Fuchs C,Siegel N,et al. Functional interaction of mammalian target of rapamycin complexes in regulating mammalian cell size and cell cycle[J]. Hum Mol Genet, 2009,18(17):3298-3310.
26. Yu Y,Yoon SO,Poulogiannis G,et al. Phosphoproteomic analysis identifies Grb10 as an mTORC1 substrate that negatively regulates insulin signaling[J]. Science,2011,332 (6035):1322-1326.
27. Agarwal R, Carey M, Hennessy B, et al. PI3K pathway-directed therapeutic strategies in cancer. Curr Opin Investig Drugs 2010; 11:615-28.
28. Foster KG,Fingar DC. Mammalian target of rapamycin (mTOR):conducting the cellular signaling symphony[J]. J Biol Chem,2010,285(19):14071-14077
29. Hennessy BT, Smith DL, Ram PT, et al. Exploiting the PI3K/AKT pathway for cancer drug discovery. Nat Rev Drug Discov 2005; 4:988-1004.
30. Karni R, de Stanchina E, Lowe SW, et al. The gene encoding the splicing factor SF2/ASF is a proto-oncogene. Nat Struct Mol Biol 2007; 14:185-93.
31. Kumar R, Hung MC. Signaling intricacies take center stage in cancer cells. Cancer Res 2005; 65:2511-5.
32. Manning BD, Cantley LC. AKT/PKB signaling: navigating downstream. Cell 2007; 129:1261-74
33.Yim EK, Peng G, Dai H, et al. Rak functions as a tumor suppressor by regulatingPTEN protein stability and function. Cancer Cell 2009; 15:304-14.
34. Marty B,Maire V,Gravier E,et al.Frequent PTEN genomic al-terations and activated phosphatidylinositol 3-kinase pathway in basal-like breast cancer cels[J].Breast Cancer Res,2008,10(6):R101.
35. Stemke-Hale K, Gonzalez-Angulo AM, Lluch A, et al. An integrative genomic and proteomic analysis of PIK3CA, PTEN, and AKT mutations in breast cancer. Cancer Res 2008; 68:6084-91
36. Barbareschi M, Buttitta F, Felicioni L, et al. Different prognostic roles of muta-tions in the helical and kinase domains of the PIK3CA gene in breast carcinomas.Clin Cancer Res 2007; 13:6064-9.
37. Aleskandarany MA,Rakha EA,Ahmed MA, et al.PIK3CA ex-presion in invasive breast cancer: a biomarker of poor prognosis [J].Breast Cancer Res Trea,2009,122(1):45-53.
38. Bachman KE, Argani P, Samuels Y, et al. The PIK3CA gene is mutated with high frequency in human breast cancers. Cancer Biol Ther 2004; 3:772-775.
39. Kirkegard T,Witon CJ,McGlynn LM,et al.AKT activation predicts outcome in breast cancer patients treated with tamoxifen[J].J Path,2005,207(2):139-146.
40. Vasudevan KM,Barbie DA,Davies MA,et al.AKT-independent ignaling downstream of oncogenic PIK3CA mutations in humancancer[J].Cancer Cel,2009,16(1):21-32.
41. Semke-Hale K.An integrative genomic andproteomic analysis of PIK3 CA,PTEN,and AKT mutations inbreast cancer [J].Cancer Res,2008,68(15): 6084-6091
42. Stal O, Perez-Tenorio G, Akerberg L, et al. Akt kinases in breast cancer and the results of adjuvant therapy. Breast Cancer Res 2003; 5:R37-44.
43. Liu H, Radisky DC, Nelson CM, et al. Mechanism of Aktl inhibition of breast cancer cell invasion reveals a protumorigenic role for TSC2. Proc Natl Acad Sci USA 2006; 103:4134-9.
44. Hutchinson JN, Jin J, Cardiff RD, et al. Activation of Akt-1 (PKB-alpha) can ac-celerate ErbB-2-mediated mammary tumorigenesis but suppresses tumor invasion. Cancer Res 2004; 64:3171-8.
45. Maroulakou IG, Oemler W, Naber SP, et al. Aktl ablation inhibits, whereas Akt2 ablation accelerates, the development of mammary adenocarcinomas in mouse mammary tumor virus (MMTV)-ErbB2/neu and MMTV-polyoma middle T transgenic mice. Cancer Res 2007; 67:167-77
46. Berns K, Horlings HM, Hennessy BT, et al. A functional genetic approach identi-fies the PI3K pathway as a major determinant of trastuzumab resistance in breast cancer. Cancer Cell 2007; 12:395-402.
47. Couch FJ, Wang XY, Wu GJ, et al. Localization of PS6K to chromosomal region 17q23 and determination of its amplification in breast cancer. Cancer Res 1999; 59:1408-1411.
48. Barlund M, Forozan F, Kononen J, et al. Detecting activation of ribosomal protein S6 kinase by complementary DNA and tissue microarray analysis. J Natl Cancer Inst 2000; 92:1252-1259.
49. Barlund M, Monni O, Kononen J, et al. Multiple genes at 17q23 undergo amplification and overexpression in breast cancer. Cancer Res 2000; 60: 5340-5344
50. Van der Hage JA, van den Broek LJ, Legrand C, et al. Overexpression of P70 S6 kinase protein is associated with increased risk of locoregional recurrence in node-negative premenopausal early breast cancer patients. Br J Cancer 2004; 90: 1543-1550
51. Walker EH, Pacold ME, Perisic O, et al. Structural determinants of phosphoinositide 3-kinase inhibition by wortmannin, LY294002, quercetin, myricetin, and staurosporine. Mol Cell 2000; 6:909-919
52. Mallucci L, Wells V, Danikas A, Davies D. Turning cell cycle controller genes into cancer drugs. A role for an antiproliferative cytokine (betaGBP). Biochem Pharmacol 2003; 66:1563-1569.
53.Markman B, Serra V, Scaltriti M, et al. Pharmacodynamic (PD) endpoints and cellular proliferation effects of NVP-BEZ235, a dual PI3K/mTOR inhibitor, in breast cancer cells and xenografts with wild-type (WT) and activating PI3K mutations. ASCO Annual Meeting. Clin Oncol 2008; 26:14508.
54. Kondapaka SB, Singh SS, Dasmahapatra GP, Sausville EA, Roy KK. Perifosine, a novel alkylphospholipid, inhibits protein kinase B activation. Mol Cancer Ther 2003; 2:1093-1103.
55. Hennessy BT, Lu Y, Poradosu E, et al. Pharmacodynamic markers of perifosine efficacy. Clin Cancer Res 2007; 13:7421-7431.
56. Castillo SS, Brognard J, Petukhov PA, et al. Preferential inhibition of Akt and killing of Akt-dependent cancer cells by rationally designed phosphatidylinositol ether lipid analogues. Cancer Res 2004; 64:2782-2792.
57. Yang L, Dan HC, Sun M, et al. Akt/protein kinase B signaling inhibitor-2, a selective small molecule inhibitor of Akt signaling with antitumor activity in cancer cells overexpressing Akt. Cancer Res 2004; 64:4394-4399.
58. Wiederrecht GJ, Sabers CJ, Brunn GJ, et al. Mechanism of action of rapamycin:New insights into the regulation of G1-phase progression in eukaryotic cells. Prog Cell Cycle Res 1995; 1:53-71.
59. Thomas GV, Tran C, Mellinghoff IK, et al. Hypoxia-inducible factor determines sensitivity to inhibitors of mTOR in kidney cancer. Nat Med 2006; 12:122-7.
60. Phung TL, Ziv K, Dabydeen D, et al. Pathological angiogenesis is induced by sustained Akt signaling and inhibited by rapamycin. Cancer Cell 2006; 10:159-7
61. Sarbassov DD, Ali SM, Sengupta S, et al. Prolonged rapamycin treatment inhibits mTORC2 assembly and Akt/PKB. Mol Cell 2006; 22:159-68.
62. Stoeltzing O, Meric-Bernstam F, Ellis LM. Intracellular signaling in tumor and endothelial cells:The expected and, yet again, the unexpected. Cancer Cell 2006; 10:89-91
63.Mondesire WH, Jian W, Zhang H, et al. Targeting mammalian target of rapamycin synergistically enhances chemotherapy-induced cytotoxicity in breast cancer cells. Clin Cancer Res 2004; 10:7031-42
64. Johnston SR. Enhancing the efficacy of hormonal agents with selected targeted agents. Clin Breast Cancer 2009; 9(suppl 1):S28-36.
65. Johnston SR. Enhancing the efficacy of hormonal agents with selected targeted agents. Clin Breast Cancer 2009; 9(suppl 1):S28-36.
66. Perez-Tenorio G, Stal O. Activation of AKT/PKB in breast cancer predicts a worse outcome among endocrine treated patients. Br J Cancer 2002; 86:540-5
67. Boulay A, Zumstein-Mecker S, Stephan C, et al. Antitumor efficacy of intermit-tent treatment schedules with the rapamycin derivative RAD001 correlates with prolonged inactivation of ribosomal protein S6 kinase 1 in peripheral blood mononuclear cells. Cancer Res 2004; 64:252-61.
68. Beeram M, Tan QT, Tekmal RR, et al. Akt-induced endocrine therapy resistance is reversed by inhibition of mTOR signaling. Ann Oncol 2007; 18:1323-8
69. Beesisteram M, Tan QT, Tekmal RR, et al. Akt-induced endocrine therapy rance is reversed by inhibition of mTOR signaling. Ann Oncol 2007; 18:1323-8
70. Baselga J, Semiglazov V, van Dam P, et al. Phase Ⅱ randomized study of neoad-juvant everolimus plus letrozole compared with placebo plus letrozole in patients with estrogen receptor-positive breast cancer. J Clin Oncol 2009; 27:2630-7。
71. Johnston SR. New strategies in estrogen receptor-positive breast cancer. Clin Cancer Res 2010; 16:1979-87
72. Loi S, Haibe-Kains B, Majjaj S, et al. PIK3CA mutations associated with gene signature of low mTORC1 signaling and better outcomes in estrogen receptor-positive breast cancer. Proc Natl Acad Sci U S A 2010; 107:10208-13
73. Shi Y, Yan H, Frost P, et al. Mammalian target of rapamycin inhibitors activate the AKT kinase in multiple myeloma cells by up-regulating the insulin-like growth factor receptor/insulin receptor substrate-1/phosphatidylinositol 3-kinase cascade. Mol Cancer Ther 2005; 4:1533-40
74. Wan X, Harkavy B, Shen N, et al. Rapamycin induces feedback activation of Akt signaling through an IGF-1R-dependent mechanism. Oncogene 2007; 26:1932-40.
75. Meric-Bernstam F, Gonzalez-Angulo AM. Targeting the mTOR signaling network for cancer therapy. J Clin Oncol 2009; 27:2278-87.
76. Hoeflich KP, O'Brien C, Boyd Z, et al. In vivo antitumor activity of MEK andphosphatidylinositol 3-kinase inhibitors in basal-like breast cancer models. Clin Cancer Res 2009; 15:4649-64.
77. Saal LH, Gruvberger-Saal SK, Persson C, et al. Recurrent gross mutations of thePTEN tumor suppressor gene in breast cancers with deficient DSB repair. NatGenet 2008; 40:102-7.79.
78.Mirzoeva OK, Das D, Heiser LM, et al. Basal subtype and MAPK/ERK kinase(MEK)-phosphoinositide 3-kinase feedback signaling determine susceptibility ofbreast cancer cells to MEK inhibition. Cancer Res 2009; 69:565-72
79. Wang X, Sun SY, et al. Enhancing mTOR-targeted cancer therapy. Expert Opinion on Therapeutic Targets, 2009,13(10):1193-1203,2009.
80. Lopiccolo J, Blumenthal GM, Bernstein WB, et al. Targeting the PI3K/Akt/mTOR pathway: effective combinations and clinical considerations. Drug Resistance Updates, 2008,11(1-2):32-50
81. Chiang GG, Abraham RT. Targeting the mTOR signaling network in cancer," Trends in Molecular Medicine, 2007,13(10):433-442,.
82. Abraham RT, Gibbons JJ. The mammalian target of rapamycin signaling pathway: twists and turns in the road to cancer therapy. Clinical Cancer Research, 2007, 13(11):3109-3114.
83. Choo AH, Blenis, J. Not all substrates are treated equally Implications for mTOR, rapamycin-resistance and cancer therapy," Cell Cycle, 2009,8(4): 567-572.
84. Schalm SS, J. Blenis. Identification of a conserved motif required for mTOR signaling. Current Biology, 2002,12(8):632-639.
85. Schalm S, Fingar, DF. Sabatini M, et al. TOS motif-mediated raptor binding regulates 4E-BP1 multisite phosphorylation and function. Current Biology, 2003,13(10):797-806
86. Wang X, Yue SY, Fu H, et al. Enhancing mammalian target of rapamycin (mTOR)-targeted cancer therapy by preventing mTOR/raptor inhibition-initiated, mTOR/rictor-independent Akt activation," Cancer Research, 2008, 68(18):7409-7418,
87. Wang X, Sun YH, Enhancing mTOR-targeted cancer therapy. Expert Opinion on Therapeutic Targets, 2008,13(10):1193-1203.
88. Jansen AP, Camalier CE, Stark C,et al. Characterization of programmed cell death 4 in multiple human cancers reveals a novel enhancer of drug sensitivity. Mol Cancer Ther, 2004,103:177-185
89. Afonia O, Juste D, Dan S, et al. Induction of PDCD4 tumor suppressor gene expression by RAR agonists, antiestrogen and HER-2/neu antagonist in breast cancer cells. Evidence for a role in apoptosis. Oncogene, 2004,23:8135-8157
90. Palamarchuk A, Efanov A, Maximov V, et al. Akt Phosphorylates and Regulates Pdcd4 Tumor Suppressor Protein[J].CancerResearch,2005,65(24):11282-11286.
91.Woodard J, Sassano A, Hay N, et al. Statin-dependent suppression of the Akt/mammalian target of rapamycin signaling cascade and programmed cell death 4 up-regulation in renal cell carcinoma[J]. Clin Cancer Res,2008,14(14):4640-4649.
1.朱冬兵,陈莉.乳腺癌C-erbB-2、nm23基因表达与免疫活性细胞反应的相关性[J].中国肿瘤,2007,16(6)：439441.
2. Grey SR,Dlay SS,Leone BE,et al.Prediction of nodal spread of breast cancer by using artificial neural network-based analyses of S100A4,nm23 and steroid receptor expression.Clin ExpMetastasis,2003,20(6):507-514.
3. HabashyHO, Powe DG, Rakha EA, et a.l Forkhead-box A1(FOXA1) expression in breast cancer and its prognostic signifi-cance[J].Eur JCancer, 2008,44(11): 1541-1551.
4. Wolf I, Bose S,W illiamson EA, et a.l FOXA1:growth inhibitor and a favorable prognostic factor in human breast cancer[J]. Int J Cancer, 2007,120(5): 1013-1022.
5. Sorlie T.Molecular portraits of breast cancer: tumour subtypes asdistinctdisease entities[J]. Eur J Cancer, 2004,40(18):2667-2675
6. Sotiriou C,W irapati P, Loi S, et a.l Gene expression profiling inbreast cancer: understanding themolecular basis ofhistologic gradeto improve prognosis[J]. JNatlCancer Inst,2006,98(4):262-272.
7. Jakowlew SB. Transforming growth factor-beta incancer and metastasis[J].Cancer Metastasis Rev, 2006,25(3):435-457.
8. Galliher AJ, Neil JR, Schiemann WP. Role of transforming growth factor-beta in cancer progression[J]. Future Onco,l 2006,2(6):743-763
9. Marchetti P, Cannita K,Ricevuto E,et al. Prognostic value of p53 molecular status in high-risk primary breast cancer.Ann Oncol,2003,14(5):704-708.
10. Poelman SM, Heimann R, Fleming GF, et al. Invariant p53 immunostaining in primary and recurrent breast cancer.Eur J Cancer,2004,40(l):28-32.
11.Linjawi A, Kontogiannea M, Halwani F, et al. Prognostic significance of p53,bcl-2,and Bax expression in early breast cancer. J Am Coll Surg, 2004,198(1):83-90.
12. Shibahara K, Asano M, Ishida Y, et al. Isolation of a novel mouse gene MA-3 that is induced upon programmed cell death. Gene, 1995,166(2):297-301
13. Onishi Y, Hashimoto S, Kizaki H, et al. Cloning of the TIS gene suppressed by topoisomerase inhibitors. Gene, 1998,215(2):453-459
14. Azzoni L, Zatsepina O, Abede B, et al. Differential transcriptional regulation of CD161 and a novel gene, 197/15a, by IL-2,IL-15, and IL-12 in NK and T cells. J Immunal,1998; 161;3493-3498
15. Zhang Z, Kubois RN. Detection of differentially expressed genes in human colon carcinoma cells treated with a selective COX-2 inhibition Oncogene,2001, 20:44504458
16. Schlichter U, Burk O, Worpenberg C, et al. The chicken PDCD4 gene is regulated by v-Myb. Oncogene, 2001,20:231-236
17. Cmarik J L, Min H Z, Hegamyer G, et al. Differentially expressed protein Pdcd4 inhibits tumor prmoter-induced neoplastic transformation. Proc Natl Acad Sci USA,1999,96 (24):14037-14042
18. Jansen AP, Camalier CE, Stark C,et al. Characterization of programmed cell death 4 in multiple human cancers reveals a novel enhancer of drug sensitivity. Mol Cancer Ther, 2004,103:
19. Halliard A, Halliard B, Zheng SJ, et al. Translational regulation of autoimmune inflammation and lymphoma genesis. J Immunol,2006,177:8095-902
20. Gao F, Wang X, Zhu F, et al. PDCD4 gene silencing in gliomas is associated with 5'CpG island methylation and unfavourable prognosis[J]. Journal of Cellular and Molecular Medicine,2009,13(10):4257-4267.
21. Chen Y, Knosel T, Kristiansen G, et al. Loss of PDCD4 expression in human lung cancer correlates with tumour progression and prognosis[J]. J Pathol,2003,200:640-646.
22. Li X, Xin S, Yang D, et al. Down-regulation of PDCD4 expression is an independent predictor of poor prognosis in human renal cell carcinoma patients[J]. J Cancer Res Clin Oncol,2012,138(3):529-535.
23. Wen Y H, Shi X, Chiriboga L, et al. Alterations in the expression of PDCD4 in ductal carcinoma of the breast[J]. Oncol Rep,2007,18(6):1387-1393.
24. Asangani I A, Rasheed S A, Nikolova D A, et al. MicroRNA-21 (miR-21) post-transcriptionally downregulates tumor suppressor Pdcd4 and stimulates invasion, intravasation and metastasis in colorectal cancer[J]. Oncogene,2008,27:2128-2136.
25. Motoyama K, Inoue H, Mimori K, et al. Clinicopathological and prognostic significance of PDCD4 and microRNA-21 in human gastric cancer[J]. Int J Oncol,2010,36:1089-1095.
26. Ma G, Guo K J, Zhang H, et al. [Expression of programmed cell death 4 and its clinicopathological significance in human pancreatic cancer][J]. Zhongguo Yi Xue Ke Xue Yuan Xue Bao,2005,27(5):597-600.
27. Zhang H, Ozaki I, Mizuta T, et al. Involvement of programmed cell death 4 in transforming growth factor-betal-induced apoptosis in human hepatocellular carcinoma[J]. Oncogene,2006,25(45):6101-6112.
28. Reis P, Tomenson M, Cervigne N, et al. Programmed cell death 4 loss increases tumor cell invasion and is regulated by miR-21 in oral squamous cell carcinoma[J]. Molecular Cancer,2010,9(1):238-243.
29. Wei N A, Liu S S, Leung T H, et al. Loss of Programmed cell death 4 (Pdcd4) associates with the progression of ovarian cancer[J]. Mol Cancer,2009,8:70-79.
30. Afonia O, Juste D, Dan S, et al. Induction of PDCD4 tumor suppressor gene expression by RAR agonists, antiestrogen and HER-2/neu antagonist in breast cancer cells. Evidence for a role in apoptosis. Oncogene, 2004,23:8135-8157
31. Wen Y H, Shi X, Chiriboga L, et al. Alterations in the expression of PDCD4 in ductal carcinoma of the breast[J]. Oncol Rep,2007,18(6):1387-1393
32. Chen Y, Knosel T, Kristiansen G, et al. Loss of PDCD4 expression in human lung cancer dorrelates with tumour progression and prognosis. J Pathol,2003, 200:640-655
33. Ma G, Guo K J, Zhang H, et al. [Expression of programmed cell death 4 and its clinicopathological significance in human pancreatic cancer][J]. Zhongguo Yi Xue Ke Xue Yuan Xue Bao,2005,27(5):597-600.
34. Yang H S, Jansen A P, Komar A A, et al. The transformation suppressor Pdcd4 is a novel eukaryotic translation initiation factor 4A binding protein that inhibits translation[J]. Mol Cell Biol,2003,23(1):26-37.
35. Yang H S, Matthews C P, Clair T, et al. Tumorigenesis suppressor Pdcd4 down-regulates mitogen-activated protein kinase kinase kinase kinase 1 expression to suppress colon carcinoma cell invasion[J]. Mol Cell Biol,2006, 26:1297-1306
36. Leupold J H, Yang H S, Colburn N H, et al. Tumor suppressor Pdcd4 inhibits invasion/intravasation and regulates urokinase receptor (u-PAR) gene expression via Sp-transcription factors[J]. Oncogene,2007,26:4550-4562
37. Yang HS, Knies JL, Stark C, et al. PDCD4suppresses tumor phenotype in JB6 cells by inhibiting AP-1 transactivation. Oncogene,2003,22:3712-3726
38. Nieves-Alicea R, Colburn N H, Simeone A M, et al. Programmed cell death 4 inhibits breast cancer cell invasion by increasing tissue inhibitor of metalloproteinases-2 expression[J]. Breast Cancer Res Treat,2009,114(2): 203-209.
39. Bitomsky N, Wethkamp N, Marikkannu R, et al. siRNA-mediated knockdown of Pdcd4 expression causes upregulation of p21(Wafl/Cipl) expression[J]. Oncogene,2008,27(35):4820-4829
40. Jin H, Kim T H, Hwang S K, et al. Aerosol delivery of urocanic acid-modified chitosan/programmed cell death 4 complex regulated apoptosis, cell cycle, and angiogenesis in lungs of K-ras null mice[J]. Mol Cancer Ther,2006,5:1041-1049.
41. Effects of chemotherapy and hormonal therapy for early breast cancer on recurrence and 15-year survival:an overview of the randomised trials[J]. Lancet,2005,365(9472):1687-1717.
42. Asangani I A, Rasheed S A, Nikolova D A, et al. MicroRNA-21 (miR-21) post-transcriptionally downregulates tumor suppressor Pdcd4 and stimulates invasion, intravasation and metastasis in colorectal cancer[J]. Oncogene, 2008,27:2128-2136
43. Chen Y, Liu W, Chao T, et al. MicroRNA-21 down-regulates the expression of tumor suppressor PDCD4 in human glioblastoma cell T98G[J]. Cancer Lett,2008,272(2):197-205.
44. Reis P, Tomenson M, Cervigne N, et al. Programmed cell death 4 loss increases tumor cell invasion and is regulated by miR-21 in oral squamous cell carcinoma[J]. Molecular Cancer,2010,9(1):238.
45. Wang J, Li Y, Wang X, et al. Ursolic Acid Inhibits Proliferation and Induces Apoptosis in Human Glioblastoma Cell Lines U251 by Suppressing TGF-β1/miR-21/PDCD4 Pathway[J]. Basic & Clinical Pharmacology & Toxicology,2012,111(2):106-112.
46. Zhang H, Ozaki I, Mizuta T, et al. Involvement of programmed cell death 4 in transforming growth factor-betal-induced apoptosis in human hepatocellular carcinoma[J]. Oncogene,2006,25(45):6101-6112
47. Dorrello N V, Peschiaroli A, Guardavaccaro D, et al. S6K1- and betaTRCP-mediated degradation of PDCD4 promotes protein translation and cell growth[J]. Science,2006,314(5798):467-471.
48. Liwak U, Thakor N, Jordan L E, et al. Tumor suppressor PDCD4 represses internal ribosome entry site-mediated translation of antiapoptotic proteins and is regulated by S6 kinase 2[J]. Mol Cell Biol,2012,32(10):1818-1829.
49. Palamarchuk A, Efanov A, Maximov V, et al. Akt Phosphorylates and Regulates Pdcd4 Tumor Suppressor Protein[J]. Cancer Research,2005,65(24):11282-11286.
50. Woodard J, Sassano A, Hay N, et al. Statin-dependent suppression of the Akt/mammalian target of rapamycin signaling cascade and programmed cell death 4 up-regulation in renal cell carcinoma[J]. Clin Cancer Res,2008, 14(14):4640-4649.
51.Carayol N, Katsoulidis E, Sassano A, et al. Suppression of Programmed Cell Death 4 (PDCD4) Protein Expression by BCR-ABL-regulated Engagement of the mTOR/p70 S6 Kinase Pathway [J]. Journal of Biological Chemistry,2008, 283(13):8601-8610.
52. Schmid T, Jansen A P, Baker A R, et al. Translation inhibitor Pdcd4 is targeted for degradation during tumor promotion[J]. Cancer Res,2008,68:1254-1260
53. Grant S. Cotargeting survival signaling pathways in cancer[J]. J Clin Invest,2008,118(9):3003-3006.
54. Zhang Z, Dubois R N. Detection of differentially expressed genes in human colon carcinoma cells treated with a selective COX-2 inhibitor[J]. Oncogene,2001, 20(33):4450-4456.
55. Schlichter U, Burk O, Worpenberg S, et al. The chicken Pdcd4 gene is regulated by v-Myb[J]. Oncogene,2001,20(2):231-239.
56.马刚,张浩,董明,等。大肠癌组织中PDCD4表达的临床病理学意义及其对预后的影响。中国现代医学杂志,2007,17(11)：1281-1286
57. CHen Y, Knosel T, Kristiansen G, et al. Loss of Pdcd4 expression in human lung cancer correlates with tumor progression and prognosis[J]. J Pathol, 2003,200(5):640-646.
58. Wei ZT, Zhang X, Wang XY, et al. Pdcd4 inhibits the malignant phenotype of ovarian cancer cells [J]. Cancer Sci, 2009,100(8):1408-1413
59. Zhang Z, DuBois RN. Detection of differentially expressed genes in human colon carcinoma cells treated with a selective COX-2 inhibitor. Oncogene 2001;20(33):4450-4456.
60. Leupold JH, Yang HS, Colburn NH, et al. Tumor suppressor Pdcd4 inhibits invasion/intravasation and regulates urokinase receptor (u-PAR) gene expression via Sp-transcription factors. Oncogene 2007;26(31):4550-4562.
61. Yang HS, Matthews CP, Clair T, et al. Tumorigenesis suppressor Pdcd4 down-regulates mitogen-activated protein kinase kinase kinase kinase 1 expression to suppress colon carcinoma cell invasion. Mol Cell Biol 2006;26(4):1297-1306.
62. Wang Q, Sun Z, Yang HS. Downregulation of tumor suppressor Pdcd4 promotes invasion and activates both beta-catenin/Tcf and AP-1-dependent transcription in colon carcinoma cells. Oncogene 2008;27(11):1527-1535.
63. Wen YH, Shi X, Chiriboga L, et al. Alterations in the expression of PDCD4 in ductal carcinoma of the breast. Oncol Rep 2007; 18(6):1387-1393
64. Gonzalez VV, Nieves AR, McMurty V, et al. Programmed cell death 4 inhibits leptin-induced breast cancer cell invasion. Oncol Rep,2012,27(3):861-866
65. Rene NA, Nancy HC, Ann MS, et al. Programmed cell death 4 inhibits breast cancer cell invasion by increasing Tissue inhibitor of Metalloproteinases-2 expression. Breast can Res,2009;114(2):203-209。
66. Palamarchuk K, Efanov A, MAXIMOV V, et al. Akt phosphorylates and regulates Pdcd4 tumor suppressor protein[J]. Cancer Re, 2005,65(24):11282-11286.
67.许传兵,徐忠华,刘照旭等。抑癌基因Pdcd4在肾透明细胞癌中的表达及生物学意义。中国现代医学杂志,2010,20(18)：2775-2778
68. Frankel LB, Christoffersen NR, Jacobsen A, et al. Programmed Cell Death 4 (PDCD4) is an important functional target of the MicroRNA miR-21 in breast cancer cells. J Biol Chem 2008;283(2):1026-1033.
1.Shibahara K, Asano M, Ishida Y, et al. Isolation of a novel mouse gene MA-3 that is induced upon programmed cell death. Gene, 1995,166(2):297-301
2.Chen Y, Knosel T, Kristiansen G, et al. Loss of PDCD4 expression in human lung cancer correlates with tumour progression and prognosis[J]. J Pathol,2003,200:640-646.
3. Wei N A, Liu S S, Leung T H, et al. Loss of Programmed cell death 4 (Pdcd4) associates with the progression of ovarian cancer[J]. Mol Cancer,2009,8:70-79
4. Zhang Z, Kubois RN. Detection of differentially expressed genes in human colon carcinoma cells treated with a selective COX-2 inhibition Oncogene, 2001,20:44504458
5.Leupold JH, Yang HS, Colburn NH, et al. Tumor suppressor Pdcd4 inhibits invasion/intravasation and regulates urokinase receptor (u-PAR) gene expression via Sp-transcription factors. Oncogene 2007;26(31):4550-4562.
6.Yang HS, Matthews CP, Clair T, et al. Tumorigenesis suppressor Pdcd4 down-regulates mitogen-activated protein kinase kinase kinase kinase 1 expression to suppress colon carcinoma cell invasion. Mol Cell Biol 2006;26(4):1297-1306.
7. Wang Q, Sun Z, Yang HS. Downregulation of tumor suppressor Pdcd4 promotes invasion and activates both beta-catenin/Tcf and AP-1-dependent transcription in colon carcinoma cells. Oncogene 2008;27(11):1527-1535.
8. Wen Y H, Shi X, Chiriboga L, et al. Alterations in the expression of PDCD4 in ductal carcinoma of the breast[J]. Oncol Rep,2007,18(6):1387-1393.
9.Nieves-Alicea R, Colburn N H, Simeone A M, et al. Programmed cell death 4 inhibits breast cancer cell invasion by increasing tissue inhibitor of metalloproteinases-2 expression[J]. Breast Cancer Res Treat,2009,114(2): 203-209.
10. Jansen AP, Camalier CE, Stark C,et al. Characterization of programmed cell death 4 in multiple human cancers reveals a novel enhancer of drug sensitivity. Mol Cancer Ther,2004,103-109
11. Afonia O, Juste D, Dan S, et al. Induction of PDCD4 tumor suppressor gene expression by RAR agonists, antiestrogen and HER-2/neu antagonist in breast cancer cells. Evidence for a role in apoptosis. Oncogene, 2004,23:8135-8157
12. Palamarchuk A, Efanov A, Maximov V, et al. Akt Phosphorylates and Regulates Pdcd4 Tumor Suppressor Protein[J].CancerResearch,2005,65(24):11282-11286
13.Woodard J, Sassano A, Hay N, et al. Statin-dependent suppression of the Akt/mammalian target of rapamycin signaling cascade and programmed cell death 4 up-regulation in renal cell carcinoma[J]. Clin Cancer Res,2008,14(14): 4640-4649
14. Foulkes WD, Smith IE, Reis-Filho JS. Triple-negative breast cancer [J]. N Engl J Med,2010,363(20):1938-1948.
15.O'Shaughnessy J, Osborne C, Pippen JE, et al. Iniparib plus chemotherapy in metastatic triple-negative breast cancer[J]. N Engl J Med,2011,364(3):205-214.
16. Metzger-Filho O, Tutt A, de Azambuja E, et al. Dissecting the heterogeneity of triple-negative breast cancer[J]. J Clin Oncol,2012,30(15):1879-1887.
17. Millikan RC, Newman B, Tse CK, et al. Epidemiology of basal-like breast cancer[J]. Breast Cancer Res Treat,2008,109:123-139.
18. Dent R, Hanna WM,Trudeau M, et al. Pattern of metastatic spread in triple-negative breast cancer [J]. Breast Cancer ResTreat,2009,115(2):423-428
19. Bauer KR, Brown M, Cress RD, et al. Descriptive analysis of estrogen receptor(ER)-negative, progesterone receptor(PR)-negative, and HER2-negative invasive breast cancer, the so-called triple-negative phenotype:a population-based study from the California Cancer Registry[J]. Cancer,2007, 109(9):1721-1728.
20. Kim MJ, Ro JY, Ahn SH, et al. Clinicopathologic significane of the basal-like subtype of breast cancer:A conparison with hormone receptor and Her2/ neuover expressing phenotypes[J]. Hum Pathol,2006,37(9):1217-1226
21. Sara M, Bando Y, Takahashi M, et al. Screening for basal marker expression is necessary for decision of the rapeutic strategy for triple-negative breast cancer [J]. J Surg Oncol,2008,97(1):30-34.
22. LiuZB, Liu G Y, Yang W R, et al. Triple-negative breast cancer types exhibit a distinct poor clinical characteristic in lymph node-negative Chinese patients[J]. Oncol Rep,2008,20(4):987-994.
23. Rakha EA, El-Sayed ME, Green AR, et al. Prognostic markers in triple-negative breast cancer[J]. Cancer,2007,109(1):25-32
24. Musgrove EA, Sutherland RL. Biological determinants of endocrine resistance in breast cancer. Nat Rev Cancer 2009;9:631-43
25. Cui X, Zhang P, Deng W, et al. Insulin-like growth factor-I inhibits progesterone receptor expression in breast cancer cells via the phosphatidylinositol 3-kinase/Akt/mammalian target of rapamycin pathway:progesterone receptor as a potential indicator of growth factor activity in breast cancer. Mol Endocrinol 2003; 17:575-88.
26. Ellis MJ, Ding L, Shen D, Luo J, Suman VJ, et al. Whole-genome analysis informs breast cancer response to aromatase inhibition. Nature,2012,486: 3532360
27. Stephens PJ, Tarpey PS, Davies H, Van Loo P, Greenman C, et al. The landscape of cancer genes and mutational processes in breast cancer. Nature,2012,486:4002404.
28. TCGA. Comprehensive molecular portraits of human breast tumours. Nature,2012, 490:61270.
29.Kalinsky K, Heguy A, Bhanot UK, Patil S, Moynahan ME (2011) PIK3CA mutations rarely demonstrate genotypic intratumoral heterogeneity and are selected for in breast cancer progression. Breast Cancer Res Treat 129:6352643
30. Crowder RJ, Phommaly C, Tao Y, et al. PIK3CA and PIK3CB inhibition produce synthetic lethality when combined with estrogen deprivation in estrogen receptor-positive breast cancer. Cancer Res,2009,69:3955-3962.
31. Yu K, Toral-Barza L, Discafani C, et al. mTOR, a novel target in breast cancer: the effect of CCI-779, an mTOR inhibitor, in preclinical models of breast cancer. Endocr Relat Cancer 2001; 8:249.
32. Simoncini T, Hafezi-Moghadam A, Brazil DP, et al. Interaction of oestrogen receptor with the regulatory subunit of phosphatidylinositol-3-OH kinase. Nature 2000;407:538.
33. Clemens, M. J., and Bommer, U. A. Translational control:the cancer connection. Int. J. Biochem. Cell Biol.,31:1-23,1999.
34. Dufner, A., Andjelkovic, M., Burgering, B. M., Hemmings, B. A., and Thomas, G. Protein kinase B localization and activation differen-tially affect S6 kinase 1 activity and eukaryotic translation initiation factor 4E-binding protein 1 phosphorylation. Mol. Cell. Biol.,19:4525-4534,1999
35. Peterson, R. T., Desai, B. N., Hardwick, J. S., and Schreiber, S. L. Protein phosphatase 2A interacts with the 70-kDa S6 kinase and is activated by inhibition of FKBP12-rapamycin-associated protein. Proc. Natl. Acad. Sci. USA, 96:4438-4442,1999
36. Jefferies, H. B., Fumagalli, S., Dennis, P. B., Reinhard, C., Pearson, R. B., and Thomas, G. Rapamycin suppresses 5□TOP mRNA translation through inhibition of p70s6k. EMBO J.,16:3693-3704,1997
37. Sekulic, A., Hudson, C. C., Homme, J. L., Yin, P., Otterness, D. M., Karnitz, L. M., et al. A direct linkage between the phosphoinositide 3-kinase-AKT signaling pathway and the mammalian target of rapamy-cin in mitogen-stimulated and transformed cells. Cancer Res., 60:3504-3513,2000
38. Reynolds, T. H. T., Bodine, S. C., and Lawrence, J. C., Jr. Control of Ser2448 phosphorylation in the mammalian target of rapamycin by insulin and skeletal muscle load. J. Biol. Chem.,277:17657-17662,2002.
39. Inoki, K., Li, Y., Zhu, T., Wu, J., and Guan, K. L. TSC2 is phosphorylated and inhibited by Akt and suppresses mTOR signalling. Nat. Cell Biol.,4:648-657, 2002.
40. Weng, Q. P., Kozlowski, M., Belham, C., Zhang, A., Comb, M. J., and Avruch, J. Regulation of the p70 S6 kinase by phosphorylation in vivo. Analysis using site-specific anti-phosphopeptide antibodies. J. Biol. Chem., 273:16621-16629, 1998
41. Sarbassov DD, Ali SM, Kim DH et al. Rictor, a novel binding partner of mTOR, defines a rapamycin-insensitive and raptor-independent pathway that regulates the cytoskeleton. Curr Biol 2004;14:1296-1302.
42. Huang J, Dibble CC, Matsuzaki M et al. The TSC1-TSC2 complex is re-quired for proper activation of mTOR complex 2. Mol Cell Biol 2008;28:4104-4115.
43. Sarbassov DD, Guertin DA, Ali SM et al. Phosphorylation and regulation of Akt/PKB by the rictor-mTOR complex. Science 2005;307:1098-1101
44. Walsh S, Flanagan L, Quinn C, et al. mTOR in breast cancer: differential expression in triple-negative and non-triple-negative tumors. Breast 2012, 21(2):178-182.
45. Shir-Hwa U, Shin-Cheh C, Yu-Sun Chang, et al. Phosphorylated mTOR expression correlates with poor outcome in early-stage triple negative breast carcinomasInt J Clin Exp Pathol 2012;5(8):806-813
46. Kuo CJ, Chung J, Fiorentino DF et al. Rapamycin selectively inhibits in-terleukin-2 activation of p70 S6 kinase. Nature 1992;358:70-73
47. Meric-Bernstam F, Gonzalez-Angulo AM. Targeting the mTOR signaling network for cancer therapy. J Clin Oncol 2009;27:2278-2287
48. LearyA,DowsettM.Combinationtherapywitharomataseinhibitors:the next era of breast cancer treatment? Br J Cancer 2006;95:661-666
49. SunSY,RosenbergLM,WangXetal.ActivationofAktandeIF4Esur-vival pathways by rapamycin-mediated mammalian target of rapamycin inhibition. Cancer Res 2005;65:7052-7058.
50. Lindsley CW. The Akt/PKB family of protein kinases:a review of small molecule inhibitors and progress towards target validation:a 2009 update. Curr Top Med Chem 2010;10:458-477
51,Ozbudak IH, Karaveli S, Simsek T,et al. Neoangiogenesisand expression of hypoxia-inducible factorlalpha, vascular endothelial growth factor, and glucose transporter-lin endom-etrioid type endometrium adenocarcinomas[J]. Gynecol Oncol,2008,108(3):603-608.
52. Li W, Petrimpol M, Molle KD,et al. Hypoxia-induced endothelial proliferation requires both mTORCland mTORC2[J].Circ Res,2007,100(1):79-872
53. Hashemolhosseini S, NagamineY, Morley SJ, et a.l Rapamycininhibition of theGl toS transition ismediated by effectson cyclinDl mRNA and protein stability [J]. J Biol Chem, 1998,273
54. Proud CG, Denton RM. Molecularmechanisms for the control oftranslation by insulin [J]. Biochem J, 1997,328(pt2):329-341
55.Tolcher AW. Novel therapeutic molecular targets for prostatecancer: themTOR signaling pathway and epidermal growth factor receptor [J]. J Uro,l 2004, 171(2pt2):S41-S43.
56. Huang S, Shu L, DillingMB, et a.l Sustained activation of theJNK cascade and rapamycin induced apoptosis are suppressed by p53/p21(Cipl) [J]. MolCel,l 2003,11(6):1491-1501.
57. Yang, H.S., Jansen, A.P., Komar, A.A., Zheng, X., Merrick, W.C., Costes, S., Lockett, S.J., Sonenberg, N. and Colburn, N.H. (2003a) The transformation suppressor Pdcd4 is a novel eukaryotic translation initiation factor 4A binding protein that inhibits translation. Mol. Cell. Biol. 23,26-37
58.]Mudduluru, G., Medved, F., Grobholz, R., Jost, C., Gruber, A., Leupold, J.H., Post, S., Jansen, A., Colburn, N.H. and Allgayer, H. (2007) Loss of programmed cell death 4 expression marks adenoma-carcinoma transition, correlates inversely with phosphorylated protein kinase B, and is an independent prognostic factor in resected colorectal cancer. Cancer 110, 1697-1707
59. Gezeh PT, Elin K, Marie AW,et al. Clinical potential of the mTOR targets S6K1 and S6K2 in breast cancer. Breast Can Res Treat,2011,128:713-723。
60. Sehgal SN. Sirolimus:its discovery, biological properties, and mechanism of action. Transplant Proc 2003;35:7S.
61. Monks A, Scudiero D, Skehan P, et al. Feasibility of a high-flux anticancer drug screen using a diverse panel of cultured human tumor cell lines. J Natl Cancer Inst1991;83:757.
62. Dancey JE. Clinical development of mammalian target of rapa-mycin inhibitors. Hematol Oncol Clin North Am 2002; 16:1101
63. Sharon BPenelope MAlexander MRapamycin Inhibits Proliferation of Estrogen-Receptor-Positive Breast Cancer Cells Journal of Surgical Research 138,37-44 (2007)
64. Yoon SO, Roux PP.Rapamycin resistance:mTORC1 substrates hold some of the answers.Curr Biol.2013 Oct 7;23(19):R880-3.
65.B.Hassan,A.Akcakanat,S.Takafumi,MTORInhibitorMLN0128hasAntitumorEffic acyinCell Lines With Intrinsic and Acquired Rapamycin-Resis-tance。 ASSOCIATION FOR ACADEMIC SURGERY AND SOCIETY OF UNIVERSITY SURGEONS; 2010
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