【作者】 陈思；

【导师】 向荣； 李娜；

【作者基本信息】 南开大学 ， 生物化学与分子生物学， 2012， 博士

【摘要】 在世界范围内，肺癌的发病率呈持续增高的趋势并且居男女肿瘤病死率的首位。据统计2009和2010年，在所有肿瘤中肺癌的发病率和病死率仍然最高[1-2]，大部分的肺癌病人在初次确诊时已经处于癌症的晚期，因此造成了肺癌的高病死率[3]。2011年美国有221,130例新发肺癌病人以及有156,940例肺癌病人死亡[4]。在病理分型上，肺癌被划分为小细胞肺癌和非小细胞肺癌（腺癌、鳞癌和大细胞肺癌），这一病理分型为治疗策略的选择和预后的评判提供参考的依据。非小细胞肺癌（NSCLC）占肺癌的比例超过85%，在初次确诊时大多数病人的原位肿瘤已经为晚期并伴有肿瘤转移，而其中肺腺癌占非小细胞肺癌约60%-70%的比例。手术切除肿瘤是对于早期非小细胞肺癌治疗的主要手段[5]，而对于不能手术切除肿瘤的病人，通常采用放疗和化疗的办法。对于小细胞肺癌而言化疗是主要的治疗方式，对一些处于特定临床分期的肿瘤也采取合并放疗的方式[5]。许多研究表明在小细胞肺癌和非小细胞肺癌中都存在肿瘤干细胞样细胞[6-26]。这些肺癌干细胞具有高克隆形成性，可以形成肿瘤并且对化疗放疗都具有抵抗性。与其他许多癌症类似，肺癌的发病也由于癌基因的激活，或抑癌基因的失活[27]。肺癌细胞的异常生长是一个多基因参与，涉及复杂的基因网络调控的过程，然而基因参与肿瘤生长并且在肺癌干细胞中的分子调控机制并不明确。因此，对肺癌分子调控机制的深入研究可提高肺癌病人的治疗质量。性别决定区域Y基因2（sex determining region Y-box2，SOX2），参与维持胚胎干细胞的多能性[28-29]，并且在多种器官发育过程中发挥作用，包括参与肺支气管的形成[30]。在肿瘤基因表达的研究中，分析在发育和分化中激活的基因结果表明，SOX2在低分化的肿瘤中高表达[31-32]。本文以SOX2为研究对象，通过各种实验手段，一方面阐明了SOX2分子在肺癌发生中的重要作用，参与调控肺癌干细胞生长及其调节的分子机制。另一方面，在观察到沉默SOX2分子可引起肺癌细胞发生显著凋亡的基础上，进而深入的研究了其调控凋亡的分子机制。这些发现为我们研制以SOX2为靶点的可应用于肺癌治疗的有效药物奠定了坚实的基础。首先,我们利用免疫组织化学染色的方法，对临床肺癌和正常肺组织标本进行了染色，分析SOX2在肺癌临床标本中的表达情况，发现SOX2在肺癌组织中的表达显著高于正常的肺组织，这一结果提示我们，SOX2在肺癌的发生中起重要的作用。其次，我们利用Hoechst33342对A549细胞进行染色并经过流式细胞仪分选，分析SOX2在人肺癌细胞A549的侧群细胞中的表达，在RNA和蛋白水平都检测出SOX2的表达在侧群细胞中明显高于非侧群细胞，说明SOX2在维持肺癌干细胞的特性中发挥了重要的作用。通过上述实验结果，我们初步明确了SOX2在肺癌发生和维持肺癌干细胞特性中的作用。我们进一步通过慢病毒系统建立了可经Doxycycline诱导沉默SOX2的稳定细胞系，并运用NOD/SCID免疫缺陷小鼠建立了肺癌体内模型，探讨了SOX2在肺癌发生和凋亡中的作用及其分子机制。主要实验结果如下：1.沉默SOX2可显著下调A549细胞中侧群细胞的比例，并且导致在侧群细胞中高表达的癌基因WNT1、WNT2、NOTCH1和c-MYC的表达量都明显下降，这一分子调控的变化在另一株人肺癌细胞系H460中可得到相同的结果。2.利用体内成像系统，连续动态的对小鼠体内肺癌细胞生长进行观察，沉默SOX2可显著抑制肺癌的发生，并且肺组织切片染色结果显示，癌基因WNT1、WNT2、NOTCH1和c-MYC在沉默SOX2实验组的肺癌组织中的表达量都明显下降。3.通过基因芯片分析，发现沉默SOX2可调控246个癌基因的表达发生变化，进一步揭示了SOX2在肺癌发生中的潜在分子机制。4.沉默SOX2可引起肺癌细胞系A549、H460和H1299细胞死亡，通过AnnexinⅤ和PI染色结果可知细胞发生了明显的凋亡。5.通过凋亡蛋白芯片分析，结合Western Blot结果验证，沉默SOX2引发的凋亡是通过调控了凋亡的线粒体途径和死亡受体途径共同作用发生的，并且SOX2调控表达变化最为显著的蛋白是p53的表达和磷酸化水平的上调、Bax和Bad分子的表达增加，caspase3和caspase8分子的激活。6. Real-time PCR和Western Blot结果显示，沉默SOX2可引起RAS/MAP激酶通路的分子表达量上调，并且激活凋亡通路中的两个重要的诱发因子p53和TNFα，并且下调Survivin的表达。7.在沉默SOX2的同时沉默RAS或MAP4K4分子或者过表达Survivin都能明显的抑制凋亡发生的程度，经AnnexinⅤ和PI染色结果可知，发生凋亡的细胞比例明显下降。8.对人肺癌组织芯片的免疫组化染色结果可知，SOX2的蛋白表达水平与Survivin的表达水平呈正相关。9.小鼠体内实验证实，沉默SOX2可引起已形成的肺肿瘤细胞发生显著的凋亡，并且引起与体外实验结果一致的p53、Survivin和TNFα表达量的变化。主要实验结论如下：1. SOX2在肺癌发生和凋亡信号通路中具有重要作用，并且可与Survivin共同作为标志物应用于肿瘤诊断。2. SOX2是通过调控肺癌侧群细胞中的癌基因的表达进而起到调控肺癌发生作用。3. SOX2调节肺癌细胞凋亡的分子机制是通过调控RAS-MAP4K4-Survivin这一下游信号通路来实现的。本文首先从观察到SOX2在肺癌组织和肺癌侧群细胞中高表达这一现象出发，并且在沉默SOX2后检测到了肺癌侧群细胞比例的下降和细胞大量凋亡现象的发生，进而阐明了SOX2调控肺癌发生和肿瘤细胞凋亡的分子机制。这一实验结果将为以SOX2为靶点的肺癌治疗策略的研发提供理论基础。更多还原

【Abstract】 Lung cancer incidence continues to rise and is the number one cause of cancerdeath in both men and women worldwide. In2009and2010, lung cancer still had thehighest incidence and mortality of all cancers [1-2]. Most cases of lung cancer arefound at an advanced stage and thus are associated with a high mortality rate [3]. In2011, there is a projected221,130new cases and156,940deaths in the United States[4]. Lung cancers are categorized as small cell carcinoma or non–small cellcarcinoma (including adenocarcinoma, squamous cell carcinoma, large cellcarcinoma). These categories division are useful for the choice of different treatmentmethods and predicting the prognosis of tumor patients. Non-small cell lung cancer(NSCLC) represents more than85%of the cases with most patients having eitherlocally advanced or metastatic disease at the time of initial diagnosis, andapproximately60%–70%of them have an adenocarcinoma histologic subtype.Surgical resection is the mainstay treatment for patients with early stage NSCLC [5].Treatment for unresectable NSCLC may involve in radiotherapy and chemotherapy.Chemotherapy (combined with radiotherapy in limited stage disease) is the mainstayof treatment for small cell lung carcinoma (SCLC)[5]. Previous study indicates thatboth SCLC and NSCLC contain stem-like cancer cells [6-26]. These lung cancer stemcells (CSCs) have been demonstrated to possess high clonogenic and tumorigenicpotential properties and generally show resistance to chemo-or radiotherapies.Similar to many other types of cancers, lung cancer is initiated by activation ofoncogenes or inactivation of tumor suppressor genes [27]. For this reason, a betterunderstanding of the molecular mechanisms of the disease will contribute toimprovement of therapeutic outcome of lung cancer patients.Sox2is a transcription factor that is involved in the maintenance of embryonicstem cell pluripotency [28-29] and in multiple developmental processes, includinglung branching morphogenesis [30]. Studies on tumor gene expression have shown that Sox2is overexpressed in certain poorly differentiated subtypes of cancer[31-32].This study applied for various experiments to analysis the roles of SOX2in lungcancer to demonstrate that SOX2contributes to the tumorigenesis of lung cancer cellsand plays an important role in maintaining the unique properties of CSCs.Furthermore, the data also indicated that silencing of SOX2gene can effectivelyinduce an apoptosis and revealed the regulatory mechanism of SOX2in apoptosissignals. Our data strongly supports the concept that specific targeting of SOX2is apromising new strategy in treatment of human lung carcinoma.The following strategies were applied for our researches:First, we performed an immunohistochemistry to analyze the level of SOX2expression between human lung tissues from normal individuals and patients bearedwith lung cancer, such as adenocarcinoma, squamous cell carcinoma, large cell andsmall cell carcinoma. We demonstrated that a specific overexpression of SOX2is inall types of lung cancer tissues. This finding suggested that SOX2contributes to thetumorigenesis of lung cancer cells.Second, dramatically higher expression of SOX2was detected in side population(SP) cells than none side population (NSP) cells of human lung adenocarcinoma cellline-A549by using Hoechst33342efflux method. This result demonstrated thatSOX2plays a vital role in maintainining the unique properties of CSCs.To further elucidate the function of SOX2in lung cancer and its regulatorymechanism, a lentivirus system was used to deliver shRNA to cultured humanNSCLC cells to establish stable cell line. Moreover, a xenografted lung cancer modelwas established in NOD/SCID mice by tail vein injection.Our major foundings are as follows:1. Significant reduced percentage of SP in A549cells with SOX2gene silencingwas revealed. And significant reduced the expression levels of WNT1, WNT2,NOTCH1and c-MYC were shown in A549cells with SOX2down-regulation. Thesame regulatory effects of SOX2on WNT1, WNT2, NOTCH1and c-MYC were alsoobserved in the human large cell lung carcinoma cell line H460with SOX2silencing. 2. The presence of A549cells in xenografted mice were monitored bynoninvasive bioluminescence imaging. We found a reduced tumorigenesis potentialof A549cells in vivo as well as a significantly attenuated expression of WNT1,WNT2, NOTCH1and c-MYC after silencing of the SOX2gene.3. More than246cancer genes regulated by SOX2were revealed by RNA-Seqarray.4. Silencing of SOX2gene in lung cancer cell lines (A549, H1299and H460)inhibited cell growth and caused significant cell death. When these cell lines werestained with Annexin V and PI, significantly increase apoptotic cells were found afterSOX2silencing.5. Proteomic array assays and Western blotting demonstrated thatdown-regulation of SOX2activates both mitochondria and death receptor signalingpathways. This was indicated by SOX2silencing resulting in marked overexpressionof p53and its phosphorylation forms, overexpression of Bax and Bad, and activationof caspase3and8in human NSCLC cells.6. It was suggested by Real-time PCR and Western blotting assay thatdown-regulation of SOX2leads to activation of the Ras/MAP4K4signaling pathwaywhich in turn, activates transcription of two key inducers of apoptosis signals-TNF-αand p53and also down-regulates expression of Survivin.7. We further showed in vitro that the apoptotic effects of SOX2silencing werecompromised by either overexpression of Survivin or down-regulation of RAS andMAP4K4by staining both Annexin V and PI.8. It was reveled that the protein expression level of SOX2closely correlatedwith that of Survivin in human lung tissues by immunohistochemical staining.9. In vivo experiment results showed that silencing of the SOX2gene inducedapoptosis. Immunohistochemical staining of the xenofrafted lung tumor tissuesrevealed the reduced protein expression levels of SOX2and Survivin, butsignificantly increased ones of p53and TNFα in shRNA-SOX2treated group whencompared to those of controls.The main conclusions are as follows: 1. SOX2plays an important role in carcinogenesis and anti-apoptotic property oflung cancer. SOX2and Survivin could be as dual-diagnosis factors to predicte thestages of clinical progression and prognosis of NSCLC patients.2. SOX2may regulate the expression of oncogenes in CSCs that promote the thedevelopment of human lung cancer.3. The RAS-MAP4K4-Survivin is one of vital signaling pathway in downstreammediated and regulated apoptosis effects induced by SOX2silencing.Taken together, our data demonstrated that the impressive level of SOX2expression was found in both human lung cancer tissue and lung cancer stem cells. Asilencing of SOX2could also reduce the percentage of SP cells and induce anapoptosis. Most important, we demonstrated insight into the regulatory mechanism ofSOX2in tumorigenesis and apoptosis. We believe that targeting of SOX2is apromising new strategy for the future treatment of human lung carcinoma.更多还原