【作者】 胡军；

【导师】 杨佩满；

【作者基本信息】 大连医科大学 ， 病理学与病理生理学， 2007， 博士

【摘要】 卵巢癌是女性生殖系统最常见的恶性肿瘤之一,且发病率在世界范围内呈上升趋势。对于卵巢癌患者,肿瘤发生了转移是致死的主要原因。肿瘤转移是一个多步骤的发展过程,肿瘤细胞从原发肿瘤上分离并且侵入到周边组织是肿瘤转移的第一步,也是最关键的一步。在正常组织中,细胞彼此间紧密连接在一起而不能自由移动;但肿瘤细胞彼此间连接松散,可以自由地与原发灶分离并迁移出去。目前研究已经明确上皮性钙黏蛋白(E-cadherin)调控此过程,该蛋白编码基因位于染色体16q22.1,蛋白分子量120kD,是一种钙依赖性跨膜糖蛋白,主要介导同质同嗜性黏附,即具有相同钙黏蛋白的细胞之间的黏附作用。在乳腺癌、食管癌、胃癌、口腔癌、肝细胞癌、甲状腺癌、膀胱癌和肺癌等肿瘤的研究中发现,E-cadherin表达降低与肿瘤的发生、分化、侵袭、转移和预后密切相关。E-cadherin的低表达使肿瘤细胞间黏附力降低,肿瘤细胞易从局部脱落,进而发生转移,故认为E-cadherin是肿瘤转移抑制因子。E-cadherin介导的细胞间黏附需要其细胞内成分与胞浆内的连环蛋白(catenin)相互作用,形成E-cadherin/ catenins复合体,并与细胞骨架相连。连环蛋白包括α-catenin (102 kD),β-catenin (92 kD)和γ-catenin (80 kD)。其中β-catenin可连接E-cadherin与α-catenin,进而与细胞骨架的肌动蛋白丝网结合。与α-catenin相比,β-catenin与E-cadherin的胞质内结构域有更直接的相互作用。有研究发现,在表达正常E-cadherin的肿瘤细胞中,β-catenin突变或缺失可致细胞解聚;转染β-catenin cDNA又可使其恢复聚集和黏附。说明β-catenin是肿瘤转移抑制基因,在细胞黏附中可独立发挥作用。β-catenin除了参与细胞间黏附外,还调控多条信号传导通路,作为Wnt信号传导通路中的关键分子而日益受到关注。当Wnt信号通路被激活,β-catenin基因突变,或大肠腺瘤样息肉复合体(APC)突变,细胞质中游离的β-catenin水平会升高并进入核内,与Tcf/Lef-1形成复合体,启动靶基因C-myc,cyclin D1,CD44,MMP-7等的复制与转录,最终导致细胞的恶性转化。细胞间的黏附除了E-cadherins/catenins复合体机制外,胞膜窖(caveolae)及窖蛋白-1(caveolin-1)也起重要作用。胞膜窖是50–100 nm的胞膜内陷,在细胞内吞和信号传导中发挥重要作用。胞膜窖的主要成分――窖蛋白-1是一22 kD的膜内在蛋白,对胞膜窖内陷形成和功能起重要作用。Caveolin-1对多种信号分子起调控作用,并与APC-β-catenin-Tcf/Lef通路之间存在交叉,与肿瘤的关系十分密切。研究表明,在受Ha-Ras,v-Abl, Myc,Neu等癌基因转化的细胞中,caveolin-1 mRNA和蛋白表达水平均下调。在乳腺癌、肺癌、结肠癌和卵巢癌等肿瘤中也观察到caveolin-1蛋白水平表达下降,细胞增殖率增加,肿瘤转移增强,提示caveolin-1在肿瘤进展中具有负调控作用。但在某些癌,如前列腺癌中,caveolin-1却呈表达增强状态,且有促进癌细胞的增殖及淋巴结转移的作用。这提示caveolin-1在肿瘤中的生物学作用具有双向性,其调节机制需进一步研究探讨。近年来,随着对肿瘤转移机制的深入研究,发现一些细胞因子参与了肿瘤细胞的转移,这些细胞因子通过与受体结合并经信号传导而引发了肿瘤细胞的运动。表皮生长因子(EGF)及其受体(EGFR)、肝细胞生长因子(HGF)及其受体(c-Met)即属于此类。EGF是首先被发现的一种多效生长因子,有刺激上皮细胞迁移、DNA合成等生物学效应,对肿瘤细胞的异常增殖也有一定作用。EGF只有与其受体EGFR结合才能发挥作用。EGFR位于胞膜窖内,大多数上皮细胞表达EGFR,其介导的信号传导诱导了内在的酪氨酸激酶活化和细胞信号,导致细胞生长和增殖。EGFR的表达增强与许多人类肿瘤的预后不良和侵袭性进展相关,但其下游靶点还不很清楚。有研究发现EGF的刺激可导致E-cadherin介导的细胞间黏附的降解,诱导肿瘤细胞的增殖和转移。肝细胞生长因子(HGF)或称分散因子(SF)是由间质细胞产生的多功能肽信号,对上皮细胞有强大的促有丝分裂、形态发生、细胞运动及血管生成等作用。其生物活性由单一受体c-Met蛋白所介导,c-Met具有酪氨酸激酶活性,主要存在于上皮细胞的细胞膜。HGF/c-Met通路促进肿瘤细胞的生存和生长,并且在血管发生和转移中起重要作用。但HGF-c-Met信号通路的作用靶分子及其与增殖、侵袭、转移联系的分子机制尚不清楚。有研究发现HGF能引起E-cadherin入胞,伴随着细胞间黏附的降解和细胞分散。综上所述,E-cadherin是重要的肿瘤转移抑制因子,而卵巢癌的早期转移是重要的死亡原因。目前,国内外对卵巢癌转移机制,特别是E-cadherin表达异常在卵巢癌发病和转移中的意义研究报道很少。本研究以人卵巢浆液性囊腺癌高、低转移细胞系HO-8910Pm和HO-8910细胞(由浙江肿瘤研究所建系,但其分子机制不明)为模型,以裸鼠为实验动物,从研究E-cadherin与卵巢癌转移的相关性入手,联系与黏附相关的信号传导分子,探讨可能的机制,以期为抗卵巢癌转移的药物设计提供新靶点。本文的主要工作如下:1.E-cadherin,β-catenin和caveolin-1基因在人卵巢浆液性囊腺癌高、低转移细胞系中的差异表达本研究首先探究了人卵巢浆液性囊腺癌高、低转移细胞系中E-cadherin,β-catenin和caveolin-1基因表达的差异,初步探讨卵巢癌转移的可能机制。应用MTT法检测细胞的增殖能力;黏附实验检测细胞与细胞外基质的黏附能力;Transwell小室法检测细胞的侵袭、迁移能力;通过免疫荧光实验、蛋白印迹实验、逆转录聚合酶链反应分别分析细胞中E-cadherin、β-catenin和caveolin-1基因在蛋白水平的表达差异、亚细胞定位及mRNA水平的表达差异。结果显示,高转移细胞系HO-8910Pm细胞的增殖能力,与细胞外基质的黏附能力,细胞的侵袭能力、迁移能力都明显高于低转移细胞系HO-8910细胞。免疫荧光实验、蛋白印迹实验表明HO-8910细胞中,E-cadherin,β-catenin和caveolin-1基因在蛋白水平的表达均较多,主要分布在细胞-细胞连接处的细胞膜上,胞质内也有少量分布;而HO-8910Pm细胞中3种基因表达都极少。逆转录聚合酶链反应证实上述表达差异是mRNA水平上的(p<0.05)。结果提示E-cadherin,β-catenin和caveolin-1基因的低表达可能与人卵巢癌侵袭和转移能力增强相关。2.RNAi技术抑制E-cadherin基因表达对HO-8910细胞生物学行为的影响为进一步确定E-cadherin的表达缺失与卵巢癌细胞的侵袭、迁移能力增强直接相关,应用RNA干扰(RNAi)技术抑制HO-8910细胞中E-cadherin表达,通过体内、外实验研究细胞增殖、侵袭、转移能力的改变。将E-cadherin siRNA转染入HO-8910细胞,特异性沉默了靶基因E-cadherin的表达。免疫荧光和蛋白印迹分析显示HO-8910/RNAi细胞中E-cadherin的蛋白水平表达与未给予RNAi处理的对照组相比明显下降;转染非特异性的control siRNA无干扰效果。说明特异性的RNAi有效。体外实验显示HO-8910/RNAi细胞的增殖、侵袭、迁移能力明显高于对照组细胞。裸鼠体内实验证明实验组肿瘤生长速度远远大于对照组,说明E-cadherin表达下调后,在体内也能促进肿瘤细胞的增殖(p<0.05)。结果提示,E-cadherin基因的部分缺失可以促进卵巢癌细胞增殖和转移。3.表皮生长因子、肝细胞生长因子对E-cadherin介导的黏附功能的调节为探讨E-cadherin的可能调节机制,研究了表皮生长因子(EGF)和肝细胞生长因子(HGF)对E-cadherin介导的黏附功能的影响。用20ng/ml EGF和10ng/ml HGF分别处理HO-8910细胞72h,HO-8910细胞的生物学行为改变经形态学方法,细胞增殖实验,体外细胞侵袭、迁移实验和划痕实验进行检测。EGF或HGF处理后的细胞E-cadherin、β-catenin和caveolin-1基因表达的改变及亚细胞定位经蛋白印记、免疫荧光实验和逆转录聚合酶链反应进行检测。结果显示,EGF和HGF分别诱导了细胞由上皮样向成纤维细胞形态转化,显著促进了细胞增殖、侵袭、迁移。EGF和HGF还明显降低了E-cadherin和β-catenin的表达,同时伴随着caveolin-1的表达降低(p<0.05)。结果提示,经由EGF/EGFR和HGF/c-Met的信号很可能是E-cadherin介导的黏附功能的重要调节者。Caveolin-1可能通过调节E-cadherin/β-catenin信号通路而抑制肿瘤转移。综上所述,我们的研究表明,E-cadherin表达降低与肿瘤侵袭、转移能力增强密切相关,是卵巢癌转移抑制因子。其黏附功能的正常发挥可能经由EGF/EGFR和HGF/c-Met两个信号传导通路负调控及caveolin-1正调控。上述信号传导分子可能成为临床抑制卵巢癌转移新的靶点,为设计预防肿瘤转移新药提供了新思路。更多还原

【Abstract】 Ovary carcinoma is one of the most frequent malignant tumors of female reproductive system, and its incidence in the world has recently been increasing. For patient with ovary carcinoma, distant metastases are the major cause of mortality. Metastasis is a multistep process. The initial and the most critical steps of metastasis include detachment of malignant cells from the primary tumor and invasion into surrounding tissue. In normal tissues, cells are tightly adhered with each other, so that they are generally not allowed to migrate freely. However, the malignant cells are more loosely associated, and can freely detach from primary tumor and migrate out.In recent years, many research work have showed that the epithelial cadherin (E-cadherin) is a very important regulator of the tumor cell adhesion. E-cadherin is a 120kD transmembrane glycoprotein coded by the E-cadherin gene located in chromosome 16q22.1. It connects epithelial cells via homotypic calcium-dependent interactions. The reduced expression of E-cadherin has been implicated in several features of tumor pathology: tumor development, cell differentiation, invasion, metastasis, and prognosis in various human cancers, such as breast, esophageal, gastric, oral, hepatocellular, thyroid, bladder, and lung cancer. Low expression of E-cadherin has been regarded as one of the main molecular events involved in dysfunction of the cell-cell adhesion system, triggering cancer invasion and metastasis. therefore, E-cadherin is regarded as the tumor metastasis suppressor.E-cadherin-mediated cell adhesion requires intracellular attachment of this glycoprotein to the actin cytoskeleton via members of the cateninfamily includingα-catenin (102 kD),β-catenin (92 kD), andγ-catenin (80 kD). It is indicated thatβ-catenin links E-cadherin toα-catenin and, consequently to the actin microfilament network of the cytoskeleton. As compared withα-catenin,β-catenin interacts with E-cadherin more directly. Many research results has suggested thatβ-catenin acts as tumor suppressor involved in cell-cell adhesion. The mutation or loss ofβ-catenin gene induced cell disaggregation independently. After being transfected withβ-catenin cDNA, tumor cells recovered aggregation and adhension. In addition to its role on cell-cell adherens junctions,β-catenin is also a transducer/transcriptional factor in signal transduction pathways. Specially,β-catenin is a critical component of the Wnt signaling system.β-catenin is to the nuclear and combines with Tcf/Lef when the cytoplasrnic level ofβ-catenin is elevated due to the activation of Wnt signaling or the mutation of APC gene,β-catenin gene etc. Then theβ-catenin-Tcf/Lef complex act as a transcription molecule which resulting in the trascription of genes such as c-myc, cyclin D1,MMP-7, CD44 etc and finally leading to the malignant transform of the cells.In addition to E-cadherin and catenins as the importment adheres mechanism, caveolae and caveolin-1 are also critical for the cell-cell adhesion. Caveolae, 50–100 nm protein-coated invaginations of the plasma membrane, play an important role in endocytosis and signal transduction. Caveolin-1, a 22 kD integral membrane protein, is a major component of caveolae and is important for the form and function of caveolae. The caveolin-1 may functionally regulate the activation of caveolae-associated signaling molecules, and have close relationship with APC-β-catenin-Tcf/Lef signals. caveolin-1 expression in mRNA and protein levels are both downregulated during cell transformation by oncogene Ha-Ras, v-Abl, Myc, Neu. Moreover, low caveolin-1 protein expression is also observed in a number of human cancers, including human breast, lung, colon, and ovarian carcinomas, suggesting a negatively regulatory role for caveolin-1 in tumor development. Consistent with these observations, the proliferation rate and tumor metastasis ability of caveolin-1-deficient cells are significantly increased. But this view has been controversial because caveolin-1 over-expression is seen in a few number of cancer such as prostate cancer that can promotetumor cells proliferation and metastasis. It has shown that caveolin-1 has bidirection characteristics in tumor biology.Recent years, along with the further research of the tumor metastasis mechanism, some cytokines have been discovered to induce the tumor cell metastasis. The cytokines is combined with its receptor to cause the tumor cell migration by signal transduction system. Cell motility is an important factor for the process of invasion and metastasis of tumors and is affected by extracellular signals such as growth factors. Epidermal growth factor (EGF) is the first discovered multieffect growth factor which can stimulate epithelial cell migration and DNA synthesis. EGF also plays an important role in over proliferation of tumor cells. EGF produces a marked effect only when combining with its receptor. The EGF receptor (EGFR) is known to localize in caveolae and are down regulated through their interaction with the caveolin-1 scaffolding domain. The EGFR is expressed in most mesenchymal and epithelial cells, and its signal transduction induces intrinsic tyrosine kinase activity finally affects cell growth and proliferation. Enhanced expression of the EGFR was related to poor prognosis and aggressive progress in many human tumors. It has been reported that EGF promotes tumor cell motility and migration. However, its downstream target related to cell motility is still unclear. Growth factors have been discovered to induce the disassembly of E-cadherin-mediated cell–cell adhesion, promote proliferation and motility of cancer cells.Hepatocyte growth factor (HGF) or scatter factor(SF)is a multifunctional peptide secreted by interstitial cell. HGF acts as a mitogen, morphogen and motogen for epithelial cells. It signals via c-Met, a tyrosine kinase receptor, which undergoes tyrosine phosphorylation and activation. HGF/c-Met pathway also contributes to survival, angiogenesis, metastasis and growth of tumor. However, it is still unclear that the downstream target of HGF/c-Met signal transduction related to cell proliferation, invasion and migration. Kamei et al reported that E-cadherin internalization in response to HGF was accompanied by the disruption of cell-cell adhesion and scattering of cells.In conclusion, E-cadherin is the important tumor metastasis suppressor. Distant metastases of ovary carcinoma are the major cause of mortality.However, there are few reports describing the molecular mechanism of ovary carcinoma metastasis, especially the interrelationship between E-cadherin and ovary carcinoma metastasis. In this study, we used human ovary serous cystadenocarcinoma cell lines, HO-8910Pm and HO-8910 with high and low metastatic potential respectively (established by Zhejiang Cancer Institute, Hangzhou, China) and nude mice as experimental models to confirm the roles of expression level of E-cadherin on metastasis of ovary carcinoma and signal transduction molecules regulating cell adhesion. This experiment will supply a new target of drug design for anti-metastasis of ovary carcinoma.The main reseach work is followings:1. Experimental study on the different expression of E-cadherin,β-catenin and caveolin-1 between both high metastasatic HO-8910Pm cell line and low metastatic HO-8910 cell line.First, this study explored the role of expression of E-cadherin,β-catenin and caveolin-1 in both high and low metastatic HO-8910Pm and HO-8910 cells on metastasis of tumor cells. MTT assay and adhesion assay were used to detect the cancer cells proliferation ability and adhesion ability to ECM Gel. The cell invasion and migration abilities were assessed by using Transwell chambers. The expression of E-cadherin,β-actin and caveolin-1 in the protein and mRNA level was detected by immunofluorescence staining analyses, Western blotting analyses and RT-PCR. The results showed that the abilities of cells proliferation, adhesion, invasion and migration in HO-8910Pm cells were significantly higher than that of HO-8910 cells. The expressions of E-cadherin,β-catenin and caveolin-1 were seen mainly in HO-8910 cells but were difficult to detect on HO-8910Pm cells either at protein level or at mRNA level (p<0.05). These data indicated that the low expression of E-cadherin,β-actin and caveolin-1 may be correlated with the increased invasion and metastasis abilities of human ovary carcinoma.2. Effects of RNAi-mediated gene silencing of E-cadherin expression on thebiologic behaviors of HO-8910 cellsTo establish whether the loss of E-cadherin was responsible for increased invasive and migratory capabilities of ovary carcinoma cells, HO-8910 cells were transfected with E-cadherin siRNA to silence theexpression of the target gene. Immunofluorescence staining and Western blotting analyses showed that the expression level of E-cadherin was significantly reduced in experimental cells, comparing with control cells. And the cells transfected with control siRNA can not silence the expression of target gene. The above results suggested that the RNAi was effective. The further experiment in vitro showed that the proliferatiive,invasive and migratory capability of HO-8910/RNAi cells were all higher than that of the controls. Furthermore, the effect of E-cadherin down-regulation of HO-8910 cells on metastasis was examined in nude mice. A significant increase was seen in mean tumor volume after E-cadherin silencing (p<0.05). These results suggested that the partial loss of E-cadherin was sufficient to promote the proliferation and metastasis of ovary carcinoma.3. Effects of EGF or HGF on E-cadherin-mediated cell-cell adhesion in HO8910 cells.To determine whether EGF or HGF affects the E-cadherin-mediated cell-cell adhesion, HO-8910 cells were treated with 20ng/ml EGF or 10ng/ml HGF for 72 h. The changes of HO8910 cells biological behaviour after treatment were estimated by morphological assay, cell proliferation assay, in vitro cell invasion assay, migration assay, and scratch wound migration assay. The expression changes of E-cadherin,β-catenin and caveolin-1 with EGF or HGF treatment were investigated by Western blotting, immunofluorescence staining, and RT-PCR analysis. The results indicated that EGF or HGF induced the epithelial-like to fibroblastoid conversion of HO8910 cell line, increased the cell proliferation, and stimulated cell invasion and migration, respectively. The low membrane expression of E-cadherin andβ-catenin was accompanied by a down-regulation of caveolin-1 expression after treatment with EGF or HGF. In addition, these ligands resulted in a lower membrane associated signal of E-cadherin,β-catenin and caveolin-1 in these cancer cells (p<0.05). These results suggest that signalling via EGF/EGFR and HGF/c-Met are likely the most important mediators of E-cadherin-mediated cell-cell adhesion in human ovary carcinoma cells. Caveolin-1 may inhibit tumor metastasis through mediating E-cadherin/β–catenin signal passageway.In conclusion, the above research work shows that E-cadherin ismetastasis suppressor of HO-8910 cells. The reduced expression of E-cadherin is closely related with tumor invasion and metastasis. E-cadherin-mediated cell-cell adhesion may be negatively regulated via EGF/EGFR and HGF/c-Met signal passageways and positively regulated by caveolin-1. These results suggest that above signal transduction molecules may become new effective targets to inhibit tumor metastasis. This will supply a new idea for designing drugs inhibiting tumor metastasis.更多还原