【作者】 张弦；

【导师】 张阳；

【作者基本信息】 大连医科大学 ， 中西医结合临床， 2013， 博士

【摘要】 背景：乳腺癌是女性最常见的恶性肿瘤，临床治疗模式多为以手术切除、全身化疗、放射治疗为主的综合治疗，但仍有相当部分患者不能够治愈。临床治疗失败的主要原因之一是浸润性乳腺癌在早期就可以发生远处转移，从而成为复发的根源。一般认为转移是继发于癌细胞局部组织侵袭和浸润之后的，而影响细胞侵袭和浸润的诸多因素，如癌细胞的增生、分离、运动都直接关系到肿瘤的转移过程。近年研究认为上皮-间质转化（Epithelial-Mesenchymal Transition，EMT）在肿瘤浸润、转移过程中发挥了重要作用。EMT是指上皮来源的癌细胞在多重信号诱导后，调控了相关基因表达，从而上皮样连接蛋白逐渐丢失，癌细胞获得间质表型的过程。癌细胞由该过程增强迁移和侵袭能力，最终能够侵入细胞周围基质发生转移。从体外实验中发现，诱导乳腺癌细胞发生EMT后，细胞的运动迁移能力明显提高，通过小鼠乳腺癌模型也证实部分癌细胞发生形态和功能改变，更容易侵入周围组织，这些基础研究都提示了EMT可能影响乳腺癌细胞的运动和侵袭能力；此外，虽然EMT对乳腺癌临床预后的影响目前尚不清楚，一些研究也显示了早期乳腺癌患者血中的肿瘤细胞（circulating tumor cells，CTC）过表达EMT标记蛋白并有着较差的预后，这些CTC也表达了干细胞的某些标记蛋白，从而提示EMT参与了癌转移并影响预后。因此，逆转EMT可能是抑制乳腺癌浸润和转移的重要方法。最新研究从天然植物中提取有效成分作为逆转药物，发现可以部分地逆转乳腺癌EMT，但多数仍停留在实验室阶段，目前国内外缺乏临床上确切有效的EMT逆转药物。榄香烯是从中药莪术中提取的有效成分，具有一定的抗肿瘤活性，在多种实体瘤中显示了临床疗效。其抗肿瘤机制十分复杂，包括直接抑制杀伤肿瘤细胞、诱导肿瘤细胞凋亡分化、抗肿瘤细胞浸润和转移等。最近的研究提示其单体成分β-榄香烯可以调控多条信号通路，参与多种激酶的磷酸化过程，如Ras/MARK/ERK和PI3K/Akt/mTOR通路。这些研究进一步阐明了β-榄香烯诱导肿瘤细胞凋亡和机体保护性自噬的抗肿瘤机制。然而，尚无研究证实β-榄香烯能够阻断/逆转乳腺癌细胞的EMT过程。本课题组在前期研究中发现β-榄香烯可以通过下调Ras/MARK/ERK信号通路促进雌激素受体α（ERα）再表达，而已知ERα可以通过转移相关蛋白3（MTA3）抑制核转录因子Snail，并与另一诱导信号——转化生长因子β（TGF-β）共同调控EMT过程。本课题拟在前期研究的基础上建立乳腺癌EMT的细胞模型，明确EMT对乳腺癌细胞的影响，并通过对乳腺癌病理组织的检测，明确EMT与乳腺癌临床病理特征及预后的关系，进一步给予β-榄香烯干预，探讨其逆转EMT作用和具体机制。目的：1.通过诱导乳腺癌细胞的EMT发生，明确其对乳腺癌细胞迁移、侵袭能力的影响；2.检测乳腺癌病理组织中EMT标记物E-钙黏蛋白（E-cadherin）和Snail的表达，明确与乳腺癌临床病理特征及预后的相关性；3.证实β-榄香烯逆转乳腺癌细胞EMT的作用，并探讨其中的具体分子机制。方法：1.培养乳腺癌细胞株MCF-7，给予TGF-β1诱导癌细胞，从细胞形态、蛋白表达鉴定EMT发生，通过划痕实验、transwell侵袭实验检测乳腺癌细胞迁移、侵袭能力的改变；2.免疫组织化学法检测103例乳腺癌患者病理组织中EMT标记物E-cadherin和Snail的表达，分析其与临床病理特征、预后之间的相关性，探讨EMT对乳腺癌临床预后的影响；3.给予β-榄香烯干预乳腺癌细胞，检测药物干预前后EMT相关蛋白表达水平的改变和侵袭功能的变化，明确β-榄香烯对乳腺癌EMT的逆转作用，通过Western免疫印迹和Real Time-PCR（RT-PCR）检测核转录因子Snail以及TGF-β/Smads信号通路蛋白的表达水平，探索其逆转机制。结果：1.通过10ng/ml TGF-β1诱导24小时，乳腺癌细胞株MCF-7形态发生梭形改变，细胞间连接消失，Western免疫印迹法检测EMT相关蛋白E-cadherin，β-catenin, N-cadherin和vimentin表达发生改变，上皮样连接蛋白E-cadherin和β-catenin表达下调，而间质样蛋白N-cadherin和vimentin表达上调，提示乳腺癌细胞发生EMT，经划痕实验和transwell侵袭实验检测细胞的迁移、侵袭能力明显提升；2.免疫组织化学法检测乳腺癌病理组织中E-cadherin和Snail的表达，同时收集103例乳腺癌患者的临床资料，分析统计两者之间的相关性，结果提示E-cadherin低表达和Snail高表达与乳腺癌临床病理特征的恶性程度正相关，且与乳腺癌患者的无病生存时间正相关；3.同时给予40μg/ml β-榄香烯和10ng/mlTGF-β1干预乳腺癌细胞，细胞形态保持铺路石样的上皮形态，EMT相关蛋白未发生表达水平改变、细胞迁移和侵袭功能亦无明显提升，证实40μg/ml β-榄香烯可以逆转乳腺癌细胞EMT；Western免疫印迹和RT-PCR结果发现核转录因子SNAI1、SNAI2等在TGF-β1诱导后出现明显上调，给予40μg/ml β-榄香烯干预后，其上调作用消失，而两者是EMT的关键因子，受TGF-β/Smads信号通路调控，在检测了Smad3信号蛋白的表达后，发现β-榄香烯可以下调磷酸化Smad3蛋白的表达，80μg/ml β-榄香烯还抑制了Smad3的mRNA水平。结论：1. TGF-β1可以诱导乳腺癌细胞发生EMT，并提升细胞运动、侵袭能力；2. EMT相关基因E-钙黏蛋白和Snail的表达与乳腺癌临床预后相关；3. β-榄香烯可以逆转乳腺癌细胞的EMT发生，其机制是通过抑制磷酸化Smad3蛋白的表达和mRNA水平，阻断TGF-β/Smads信号通路从而下调核转录因子SNAI1和SNAI2的表达，最终逆转乳腺癌细胞的EMT发生。更多还原

【Abstract】 Background: Breast cancer is the most commonly diagnosed type of cancer, thesecond leading cause of cancer-related deaths in women. Invasion and metastasis are themost lethal characteristics of breast cancer: almost all of patients die from long-termrecurrence. It is well known that several steps are necessary in cancer metastasis，suchas angiogenesis, invasion, embolism and adherence, etc. However, a growing area ofinterest is the association of cancer metastasis with epithelial-mesenchymal transition(EMT). EMT is defined by the loss of epithelial characteristics and the acquisition of amesenchymal phenotype and is essential in embryonic development and inflammation.The transformation has implications in onset of breast cancer to metastasis, andincreases evidences to support hypothesis of cancer stem cell.EMT can increase breast cancer cell abilities of migration and invasion in vivo andin vitro. Recent evidences indicate that EMT is associated to chemo-resistance andcirculating tumor cells in breast cancer. Thus, the reversal of EMT may be a potentialmethod to improve the prognosis of breast cancer patients. There are several hallmarks,such as E-cadherin, catenin (epithelial marker) and N-cadherin, fibronectin, vimentin(mesenchymal marker). Dysregulation of these proteins is provoked by transcriptionrepressors involving Snail/SNAI1, Slug/SNAI2. TGF-β signalling as well as Wnt-,Notch-, Hedgehog-, and NF-κB-dependent pathways can induce and maintain EMT,considered as potential targets on EMT reversal. However, there is a more interestingapproach to block this phenotype transition.Elemene (1-methyl-1-vinyl-2,4-diisopropenyl-cyclohexane), an active componentof herb medicine Curcuma wenyujin, is a novel anti-cancer drug. The extract ofelemene is a mixture of α-, β-and δ-elemene, with β-elemene (ELE) as the maincomponents, which accounts for60~72%of the three isoforms. The anti-cancermechanism of ELE is that (i) it induces apoptosis as well as protective autophagy in human non-small-cell lung cancer A549cells;(ii) it can down-regulate the expressionof survivin, Bcl-xL and Mta-1and induce the apoptosis of T24cells at a molecular level;(iii) it participates the regulation on phosphorylation of multiple members of the kinasefamilies inducing apoptosis in tumour cells, such as in the Ras/Raf/MEK/ERK cascadeand AKP. However, it is unknown that ELE affects EMT in human breast cancer cells.We reported that ELE up-regulated expression of the oestrogen receptor alpha(ERalpha) located breast cell membrane though repressing the acitivity of theRas/MARK/ERK pathway. A previous report showed that ERalpha inhibited theexpression of Snail though metastasis related protein3(MTA3), and regulated the EMTwith TGF-β signaling together. This study plans to establish a breast cancer EMT model,cleared the effect on breast cancer cells and to detect the breast cancer tissue analysedthe relation between EMT and clinical pathological characters. At last, we explored thereversal effect of ELE on breast cancer cells.Objective:1. To identify the effect EMT induced by TGF-β1on breast cancercells;2. To detect the relations in the expression of E-cadherin and Snail and clinicalpathological characters of breast cancer patients.3. To explore the reversal effect onbreast cancer cells EMT with ELE.Methods:1. We cultivate breast cancer cell line MCF-7and induce EMT withTGF-β1, and then identify the EMT though cell shape and protein expression’s change,and then examine the cell abilities of migration and invasion by wound healing assayand transwell assay.2. We detect the expression of E-cadherin and Snail in breastcancer tissue by immunity-histochemistry, and analysis the relation in the expression ofE-cadherin and Snail with clinical pathological characters and prognosises of breastcancer patients.3. We treat the breast cancer cell EMT model with ELE and detect thechange in protein expression and cell abilities of migration and invasion, identified thereversal effect on EMT. We explore the molecular mechanism of the reversal effectthough TGF-β/Smads signal pathway and transcription factor Snail.Results:1. Treating with10ng/ml TGF-β1for24hours, the breast cancer cell lineMCF-7changed to the fusiform shape and the disappeared cell link. We find that theexpression of E-cadherin and β-catenin were down-regulated, whereas N-cadherin andvimentin were up-regulated. The increase of cell abilities of migration and invasion wasconfirmed by wound healing assay and transwell assay.2. The immunity-histochemistryshowed the expression of E-cadherin and Snail in breast cancer tissue.103breast cancerpatients were enrolled and the relation in the expression of E-cadherin and Snail and clinical pathological characters of breast cancer patients was researched. The resultsshowed low expression of E-cadherin and high expression of Snail was related withclinical pathological malignancy characters and prognosis of breast cancer patients.3.40μg/ml ELE can blocks breast cancer cell EMT induced by TGF-β1, further ELE wasable to inhibit TGF-β1-mediated up-regulation of mRNA and protein expression ofnuclear transcription factors (SNAI1and SNAI2), in part through decreasing expressionand phosphorylation of Smad3, a central protein in TGF-β1signalling pathway.Conclusions：1. The breast cancer cells can be induced to a EMT by TGF-β1andcell abilities of migration and invasion were increased.2. The expression of E-cadherinand Snail were related to clinical prognosis of breast cancer patients.3. ELE blocksEMT induced by TGF-β1in breast cancer cells and the mechanism is inhibitingTGF-β1-mediated up-regulation of mRNA and protein expression of nucleartranscription factors (SNAI1and SNAI2), in part through decreasing expression andphosphorylation of Smad3.更多还原