【作者】 裘建明；

【导师】 曹雪涛；

【作者基本信息】 浙江大学 ， 肿瘤学， 2008， 博士

【摘要】 人磷脂酰乙醇胺结合蛋白-4(Human phosphatidylethanolamine-bindingprotein-4,hPEBP4)是我们实验室自主发现并命名的新分子,其高表达于乳腺癌、卵巢癌及前列腺癌细胞和组织。我们以前的研究集中于hPEBP4和肿瘤细胞凋亡的关系上,证明肿瘤细胞中的hPEBP4能通过抑制ERK1/2、JNK的活化以及促进Akt的激活来发挥抵抗TNFα和TRAIL诱导的凋亡的作用。为了更加全面地了解hPEBP4与肿瘤的关系,我们推测作为在肿瘤细胞相对特异性高表达的基因,hPEBP4的上调可能是肿瘤细胞适应体内环境不断进化演变的结果,也就是说hPEBP4在肿瘤细胞内相对特异性的高表达对肿瘤细胞而言究竟有什么意义呢?hPEBP4高表达于乳腺癌、卵巢癌和前列腺癌,我们从hPEBP4的表达谱入手,推测其很可能参与肿瘤细胞对微环境中的性激素的反应。在本课题中,我们选取雌激素信号通路作为研究对象,首次探讨了hPEBP4在肿瘤细胞雌激素信号通路中的作用及其机制。为了确认hPEBP4是否在雌激素通路中发挥作用,我们用报告基因检测hPEBP4对雌激素受体α(estrogen recpeptor alpha,ERα)介导的转录活性的作用。人乳腺癌细胞MCF-7高表达hPEBP4,在MCF-7细胞中成功干扰hPEBP4的表达后,我们发现细胞内报告基因荧光强度明显下降。分别用real-time PCR和Western blot检测ERα下游的两个靶基因pS2和Cyclin D,发现hPEBP4的沉默显著性地削弱了雌激素诱导的肿瘤细胞内ERα下游的pS2和Cyclin D1的表达。ERα作为转录因子激活转录是雌激素信号通路最主要和经典的部分,所以这些结果表明hPEBP4参与了雌激素信号通路的活化,该分子的沉默会显著降低乳腺癌细胞ERα通路转录活性。为了寻找hPEBP4促进ERα通路活化的机制,我们用染色质免疫共沉淀的技术观察ERα在雌二醇(E2)的刺激下与靶基因启动子ER顺势作用元件(ERE)结合的情况。结果发现,在干扰hPEBP4后,ERα受E2激活、招募并结合到ERE的数量显著减少。表明hPEBP4对ERα通路活化的调控与跟启动子结合的ERα的数量下降有关。为了进一步研究hPEBP4导致招募到ERE的ERα数量减少的机制,我们检测了干扰hPEBP4对ERα蛋白水平的影响。Western blot的结果表明,在E2刺激2h、6h后,ERα的蛋白水平呈一定程度的升高,但与干扰对照组相比,hPEBP4的干扰明显降低了ERα的表达量。考虑到蛋白酶体依赖的ERα的降解是ERα通路调控的重要机制,我们用MG132来抑制蛋白酶体的降解功能,发现加入MG132可以使ERα蛋白水平在E2的刺激下迅速升高,而且hPEBP4的干扰对于ERα上升的抑制作用消失。那么,是不是相应的ERα的转录功能也发生了类似的变化呢?我们用带ERE的报告基因检测了上述处理后的转录活性,发现MG132可以完全逆转干扰hPEBP4导致的ERα转录活性的降低。最后,我们还用瞬时转染ERα真核表达质粒的方法显著逆转了干扰组的转录活性,从而证明hPEBP4确实是通过抑制蛋白酶体对ERα的降解来促进雌激素对肿瘤细胞转录活性的激活。受到凋亡信号的刺激,hPEBP4从细胞浆溶酶体到细胞膜的转位进而调控MAPK和Akt等信号分子的活化,我们以往的工作证明这是其抗凋亡作用的重要机制。那么,是否在雌激素信号通路中hPEBP4也具有这种功能呢?于是,我们检测了E2刺激下MCF-7细胞的ERK1/2和JNK的磷酸化情况,发现E2能够激活ERK1/2和JNK,而hPEBP4的干扰则显著的增强E2的这种活化作用。相对而言,E2对Akt的活化作用较微弱,干扰hPEBP4使Akt的磷酸化水平降低。另外,细胞浆免疫共沉淀的结果也证明细胞膜ERα可以和hPEBP4发生相互作用。上述结果表明hPEBP4参与了极少数ERα介导的非经典的膜信号通路的调控。那么是否hPEBP4对ERα经典的转录活性的调控就是通过对非经典的膜信号通路的作用而实现的呢?为了验证这个假设,我们用U0126和LY294002分别阻断ERK和PI-3K信号通路,然后观察ERα的报告基因转录活性的变化。我们发现,ERK和PI-3K的阻断并不能取消hPEBP4对ERα转录激活功能的促进作用,表明hPEBP4参与对膜ERα信号通路包括MAPK和PI-3K的调控,但是后者不参与hPEBP4对经典的ERα介导的转录活性的调控。最后,我们想知道hPEBP4的这种对ERα的转录活性调控作用是否只局限于MCF-7细胞。于是,我们构建了过表达hPEBP4的稳定筛选的HeLa细胞并用瞬时转染pSG5-ERα的方法赋予该细胞对E2的反应性。结果发现与空载体对照细胞相比,过表达hPEBP4的HeLa细胞具有更强的报告基因转录活性,并且ERα蛋白表达水平更高。当加入蛋白酶体抑制剂MG132后,ERα蛋白水平的差异消失。这一结果说明,hPEBP4对ERα介导的转录功能的调控在肿瘤细胞中存在广泛性。综上所述,我们通过分别在MCF-7和HeLa细胞中RNA干扰和稳定过表达hPEBP4,证明hPEBP4能通过抑制蛋白酶体依赖的ERα的降解而显著促进ERα介导的转录活性,从而首次揭示了hPEBP4参与肿瘤细胞雌激素信号通路的活化,本实验也为ERα介导的信号通路的调控机制增加了新的内容。人磷脂酰乙醇胺结合蛋白-4(Human phosphatidylethanolamine-bindingprotein-4,hPEBP4)是一个我们实验室发现并克隆的“新基因”,在乳腺癌细胞中高水平表达,具有抵抗凋亡的作用。前期的报道已经证实了用siRNA沉默hPEBP4的表达后,乳腺癌对TNF-α的敏感性明显增强,前列腺癌对TRAIL的敏感性也有较大的提高。然而RNA干扰技术要运用于体内甚至临床还存在许多障碍,而小分子化合物作为抑制剂则具有技术平台成熟、口服即可吸收、不易降解、起效快等优点,所以我们与中科院药物所合作拟开发一个能特异性阻断hPEBP4的小分子化合物,希望能在前面机制研究的基础上做一些治疗性研究的有益探索。一、小分子化合物的发现因为目前仍缺乏一个确切的关于hPEBP4的晶体衍射的结果,所以我们根据已知的同源分子采取了同源模建的方法来建立hPEBP4分子的三维结构模型。用hPEBP4的基因登录号(AY037148)搜索PDB的同源蛋白序列(http://www.ncbi.nih.gov/BLAST/),发现三个同源分子牛PEBP-1(PDB登录号1A44),小鼠PEBP-2(登录号1KN3),人PEBP-1(登录号1BEH)。以这三个同源分子为参考,我们用多序列结构对比软件InsightⅡ结合人工调整勾勒出hPEBP4分子的最有可能的三维结构模型。最后运用Powell软件从能量角度优化并验证该模型。我们选用SPECS化合物库,对超过270000个小分子化合物逐一进行了筛选。具体的方法是先用Dock4.0将小分子逐一跟hPEBP4的3D模型进行对接,淘汰结合不太紧密的小分子,此轮筛选留下8700个小分子。然后,用另外一个叫Flex的软件对这些候选分子打分选取前600个。接着用autodock3.05进行hPEBP4和化合物的自动对接,并依据类药性原则将一些不符合药物特征的化合物淘汰,最后得到83个最佳分子进行合成。合成好的83个候选化合物先用表面等离子共振(SPR)技术筛选出确实能跟靶标蛋白hPEBP4结合的化合物。SPR是检测蛋白等生物大分子相互之间及大分子与小分子相互作用的一种生物传感器。在这里我们把融合蛋白GST-hPEBP4耦连在CM5芯片上,然后把化合物小分子溶解在溶液中以流动相流经芯片表面,根据不同浓度化合物与蛋白结合能力(近似反映为Biacore的RU值)的不同来计算平衡解离常数,从而得到两者结合性的信息。运用这种方法,我们最终拿到了7个跟hPEBP4结合最强的小分子并用MTT的方法初步观察其抑制乳腺癌细胞增值的能力。与TNF-α(20ng/mL)联合作用,只有命名为DC240016和DC240042的两个小分子化合物表现出跟TNF-α协同抑制MCF-7细胞增值的能力。由于前者单独应用即表现出明显的毒性,故最终决定用DC240042开展下面的生物学试验进一步确证其抗肿瘤功能。二、DC240042抗肿瘤活性的鉴定以前的研究已经证明hPEBP4抵抗TNF-α引起的细胞凋亡跟其与Raf-1和MEK1相互作用,进而阻断ERK1/2和JNK的活化有关。所以为了排除化合物非特异性杀伤造成MTT阳性结果的可能,我们用TNF-α刺激MCF-7细胞后用免疫印记的方法检测ERK1/2和JNK的磷酸化情况,发现TNF-α可以引ERK和JNK的磷酸化,而预先加入了DC240042的细胞ERK1/2和JNK的磷酸化程度进一步增强。免疫共沉淀发现DC240042导致hPEBP4跟Raf-1及MEK1的作用明显减弱。这些数据说明DC240042可以从分子机制上逆转hPEBP4的作用。然后,我们用R123/PI标记的方法利用流式细胞仪研究DC240042对凋亡的作用。结果显示DC240042可以显著增强MCF-7细胞对TNF-α的敏感性。再用另外一对凋亡标记探针AnnexinV/PI检测发现DC240042跟不同浓度的TNF-α(10,20,50ng/mL)联合作用都具有对后者的增敏作用。流式细胞仪的结果还显示DC240042的这种增强凋亡的作用具有浓度和时间的依赖性。接着,我们检测DC240042对不表达hPEBP4的细胞L929是否具有促凋亡作用。流式结果显示DC240042没有对L929细胞表现出增强TNF-α凋亡作用的效应。为了排除细胞之间因遗传背景差异造成的L929细胞阴性结果,我们用RNAi沉默了MCF-7细胞中hPEBP4的表达,然后观察DC240042是否仍具有上面描述的放大TNF-α凋亡效应的作用。结果显示,RNAi可以极大增强MCF-7对TNF-α的敏感性(近三倍),但hPEBP4沉默后的MCF-7细胞对DC240042不再具有反应性。上面两个试验结果说明DC240042对TNF-α的协同效应是依赖于hPEBP4的,所以DC240042是靶向于hPEBP4的小分子抑制剂。因为缺乏一个成熟的乳腺癌TNF-α体内治疗的模型,我们进一步用软琼脂克隆形成试验考察了DC240042对MCF-7生长抑制作用。结果表明,TNF-α对MCF-7的克隆形成能力具有一个明显的抑制作用,与DC240042的联合应用则进一步增强了这种抑制作用。以前我们曾经报道了hPEBP4在前列腺肿瘤细胞抵抗TRAIL引发的凋亡过程中亦起了一定的作用。所以最后我们检测了DC240042与TRAIL联合运用对LnCap细胞的凋亡诱导作用。流式细胞检测结果表明,与单独用TRAIL相比,DC240042的联用显著增强了细胞的凋亡百分比。更多还原

【Abstract】 hPEBP4 promotes ERα-mediated transactivation in cancer cells by inhibiting proteasome-dependent degradation of ERαHuman phosphatidylethanolamine-binding protein-4 (hPEBP4), a novel molecule identified by us in 2004, is highly expressed in breast cancer, ovary cancer and prostate cancer. Our previous studies about this molecule focused on the role of hPEBP4 in the apoptosis resistance of tumor cells, and we has demonstrated that hPEBP4 promotes cancer cells to resist TNFα/TRAIL-mediated apoptosis by inhibiting the activation of ERK1/2、JNK but enhancing activation of Akt. In addition to the role of this molecule in cellular apoptosis, we are wondering whether there are any other functions of this molecule relatively specifically expressed in tumor cells. We predict that high expression of hPEBP4 in tumor cells is probably the result of constant evolution of tumor cells in response to microenvironment around them. Considering that hPEBP4 is highly expressed in the restricted type of tumors including ovary, breast and prostate cancer,, we suppose that hPEBP4 may participate the cellular response to sex-steroid hormone in tumorigenesis under microenvironment. Therefore, the primary aim of this study is to investigate the role of hPEBP4 in the estrogen recpeptor alpha (ERα)-mediated transacvivation of cancer cells.In order to address the role of hPEBP4 in ERα-mediated transactivation, we firstly used reporter gene assay to determine whether hPEBP4 could affect ERα-mediated transactivation. After silencing hPEBP4 with smll RNA interferance(siRNA) in human breast cancer cells MCF-7, we observed that the luciferase activity significantly decreased, indicating hPEBP4 could promote ERα-mediated transactivation. Real-time PCR and Western blot analysis showed that the activation of endogeneous target genes of ERα, pS2 and Cyclin D1, was also greatly suppressed when hPEBP4 was silenced , further confirming that hPEBP4 could promote ERα-mediated transactivation. The transactivation activity of ERα, as a transcriptional factor, is the major component of ERα-mediated signal pathway. So, our data suggest that hPEBP4 positively regulates ERα-mediated signal pathway and silencing hPEBP4 can significantly suppress the transactivational activity of ERαin breast cancer cells.Then we want to know how hPEBP4 can affect ERα-mediated transcriptional activity, we used chromatin immunoprecipitaion(ChIP) to analyze the binding of ERαwith estrogen responsive element(ERE) after MCF-7 cells were stimulated with E2. We found that silencing hPEBP4 led to less ERαrecruited to ERE, thus suggesting that the reduced ERα-mediated tranactivation may be due to the less amount of ERαbound with ERE.To reveal the mechanism of how hPEBP4 leads to less ERαrecruited to ERE, we obsterved the ERαprotein level in MCF-7 cells 2 h and 6 h after stimulated with E2. We found that E2 stimulation induced an increase of ERαprotein and silencing hPEBP4 dramatically suppressed E2-induced increase of ERα. Given the importance of proteasome-dependent degradation in ERαturnover, we observed the effect of proteasome inhibitor MG132 on the increase of ERαprotein by hPEBP4. We found that MG132 leads to dramatic accumulation of ERαin MCF-7 cells stimulated with E2, and more importantly, the difference of ERαin MCF-7 cells between siRNA and non-siRNA control disappeared after MG132 was added to block proteasome activity. Then we ask one question: whether MG132 has the same effect on ERα-mediated transactivation accordingly? Through reporter gene assay, we showed that MG132 almost completely abolished the inhibitor effect of silencing hPEBP4 on ERα-mediated transactivation. Furthermore, we successfully reversed the decrease of ERα-mediated transactivation by transfecting pSG5-ERαplasmid into hPEBP4-silenced MCF-7 cells. These data demonstrate that hPEBP4 promotes ERα-mediated transactivation by inhibiting proteasome-dependent degradation of ERα.Our previous studies has shown that hPEBP4 can translocate from lysosome to membrane to regulate MAPK and Akt pathway, thereby resulting in the resistance of tumor cells to apoptosis induction. We wonder whether the regulation of signal pathways by hPEBP4 is also invovled in the promotion of ERα--mediated transactivation by hPEBP4. We found that E2 could activate ERK1/2 and Akt pathway in MCF-7 cells, and silencing hPEBP4 enhanced the activation of ERK1/2 but inhibited Akt activation. In addition, we confirmed the interaction between hPEBP4 and membrane-bound ERαwith immunoprecipitation using cytoplasmic protein. Since hPEBP4 could regulate ERα-mediated membrane signal pathway(non-classical pathway), we went further to confirm whether the activity is involved in the effect of hPEBP4 on ERα-mediated transactivation. To test this hypothesis, we used U0126 and LY294002 to block activation of ERK1/2 and PI-3K respectively and then we observed the transcriptional activity with reporter gene assay in MCF-7 cells. We found that both U0126 and LY294002 didn’t affect the inhibitory effect of silencing hPEBP4 on ERα-mediated transactivation. And also we didn’t observe any effect of the inhibitors on ERαprotein level as demonstrated by Western blot. The data indicate that the effect of hPEBP4 on ERα-mediated transactivation is independent of its regulation of ERK and Akt pathways.Finally, we confirmed the above effect of hPEBP4 by using HeLa cells as cell model. We established stable cell clones overexpressing hPEBP4 by transfecting HeLa cells with pcDNA3.1/His-Myc-hPEBP4 plasmids. We also transiently transfered pSG5-ERαto HeLa cells to acquire an estrogen responsiveness. We found Overexpression of hPEBP4 led to a higher transcriptional activity of ERαand increase of ERαprotein level as well. MG132 blocked the promoting effect of hPEBP4 on ERα-mediated transactivation in HeLa cells. The results suggested that promoting effect of hPEBP4 on ERα-mediated transactivation also exists in other kinds of human cancer cells.In summary, we have demonstrated that hPEBP4 can promote ERα-mediated transactivation in cancer cells by inhibiting proteasome-dependent degradation of ERα. The present study, for the first time, links hPEBP4 with estrogen-induced activity in cancer cells, thus representing a new regulatory mechanism for ERα-mediated signal pathway. Identification of A Small Molecule Inhibitor for hPEBP4 and Its Antitumor EffectHuman phosphatidylethanolamine-binding protein-4 (hPEBP4) is a novel gene identified by our lab a few years ago. Highly expressed in breast cancer cells, it has been determined to play a role in resisting apoptosis. Previous work has reported that MCF-7 and LnCap cells become more sensitive to TNF-α/TRAIL-mediated apoptosis respectively if hPEBP4 was targetedly reduced with siRNA. However, siRNA has its bottleneck when applied to in vivo. Comparatively, development of small molecule inhibitors is more mature in technology and characterized as good absorbance and bioavailability. Therefore, based on the understanding of its effect and mechanism in apoptosis resistance, we intended to discovery a specific chemical inhibitor for hPEBP4.1、Identification of small molecule inhibitor for hPEBP4Due to a lack of X-ray result of hPEBP4 protein crystal, we adopted homology modeling to Figureure out a 3D structure of hPEBP4. To do that we found out the homology proteins by searching protein data bank(http://www.ncbi.nih.gov/BLAST/). Based on those homology proteins, we used two simutation softwares of InsightII and Powell to determine the structure of hPEBP4. Then we tried to seek suitable candidates samL1 molecules from the SPECS molecule library , which contains a pool of about 27,000,0 small molecules, by applying virtual screening method. The following softwares were used consecutively during that process: Dock4.0, Flex and AutoDock3.05. As a result, the cancdidate molecules were finally reduced to 83 after virtual screening.For the remaining 83 candidate molecules, we further screened them with surface plasmon resonance(SPR). As a useful technology in drug discovery, SPR is designed to detect any interaction between two proteins or protein-small molecules based on the local tiny change of electronical flow caused by that interaction. RU is usually used to show how strong the interaction is. In this experiment, we put the recombinant GST-hPEBP4 fusion protein on CM5 Chip and made the small molecules flow past the chip. Interaction between the fusion protein and passing small molecule was measured. With SPR, we selected out 7 small moleccules which could bind with hPEBP4 fusion protein most tightly. These molecules were further subject to MTT assay to examine their effect on proliferation of MCF-7 cells. When added together with TNF-α, two candidates—DC240042, DC240016, show a synergestic effect on inhibiting MCF-7 cell proliferation. But DC240016 alone caused a dramatic cytotoxic effect on MCF-7 cells, so we performed following functional screen only with DC240042.2、Antitumor effect of DC240042We have known that hPEBP4 promotes apoptosis through interaction with Raf-1 and MEK1 and inhibiting the phosphoralation of ERK1/2 and JNK1/2, which provides us a convinent way to rule out off-target effect of DC240042. Western blot was used to detect the activation of JNK1/2 and ERK1/2. We found that incubation with 20ng/mL TNF-αfor 30 minutes could lead to obvious phosphoralation of JNK and ERK1/2. DC240042 pretreatment significantly enhanced that phosphoralation. Co-immunoprecipitation assay suggested that DC240042 could also render the hPEBP4 bind with Raf-1/MEK1 to a less extent.Then we used FACS to investigate whether DC240042 could really promote TNF-α-mediated apoptosis. Probing with R123/PI revealed that DC240042 increased the apoptosis percent caused by 20ng/mL TNF-α. Apoptosis assay with another pair of probes, Annexin V/PI, show DC240042 was synergestically effective in promoting apoptosis when combined with 10, 20 and 50ng/mL TNF-α. FACS also suggested that the effect of DC240042 was dose and time dependent.Next, we wanted to see whether the effect of DC240042 still exist in another cell line L929 that doesn’t express hPEBP4 and sensitive to TNF-α. We found that DC240042 hadn’t any effect on L929 cells. To exclude the possibility that difference other than hPEBP4 between MCF-7 and L929 lead to the different response to DC240042, we directly silenced hPEBP4 in MCF-7 cells and tested apoptosis. Consistant with previous report, hPEBP4 silence dramatically sensitized MCF-7 cells to TNF-α(almost 3 folds). However, as observed in L929 cells, DC240042 didn’t promote TNF-α-mediated apoptosis after hPEBP4 was silenced. These data imply that the effect of DC240042 is totally dependent on hPEBP4. Therefore, DC240042, as purposely designed, targets hPEBP4 to exert its effect.Since there is no flexible and widely accepted in vivo model to perform therapheutic experiments with TNF-α, we used soft-agorase clone forming assay to examine the effect of DC240042 on the anchorage-independent proliferation of MCF-7 cells. Pretreatment of DC240042 significantly enhanced the inhibitory effect of TNF-αon the growth of MCF-7 cells.Because it has been reported that hPEBP4 also plays a role in TRAIL-resistance of prostate cancer. We tested whether DC240042 was effective in LnCap cells. FACS result show that DC240042 could also sensitized LnCap cells to TRAIL-induced apoptosis.更多还原