TRB3和BCL6促进乳腺癌发生发展的作用和机制

发布时间：2018-11-12 12:01

【摘要】：乳腺癌是世界范围内女性最常见的恶性肿瘤。虽然对乳腺癌主要采取手术、放疗并辅以化疗的联合治疗方案获得很大成功,但仍有40%的乳腺癌患者在治疗后出现肿瘤复发和癌细胞转移,是导致乳腺癌患者死亡的主要原因。最近的研究证明,乳腺癌干细胞(Breast cancer stem cells, BCSCs)是乳腺癌复发、转移和抵抗治疗的关键原因。研究证明上皮一间质转化(Epithelial-mesenchymal transition,EMT)是肿瘤细胞发生侵袭转移的必经途径。另一方面,文献报道发生EMT的乳腺癌细胞呈现BCSCs的特征。本论文的目的是分别从BCSCs和EMT两个方面研究乳腺癌复发与转移的机制,寻找开发抗乳腺癌复发与转移的潜在分子药靶。大量研究证明,炎症反应可能是促进BCSCs形成与维持的关键原因,但炎症与BCSCs生成与维持的分子机制仍有待阐明。TRB3 (Tribbles homolog 3)是假性蛋白激酶成员之一,最近的研究证明,TRB3不仅与乳腺癌的不良预后呈正相关,而且促进肿瘤的侵袭和转移。本实验室前期工作发现,炎症和应激反应增加炎症损伤组织和肿瘤组织的TRB3表达。因此我们提出假说,TRB3介导炎症反应引起的BCSCs生成及维持。检测乳腺癌组织芯片我们发现乳腺癌组织中炎症因子IL6、TRB3和转录因子Sox2的表达呈正相关。利用磁珠分选技术,我们发现由IL6诱导的CD44+/CD24- BCSCs田胞群TRB3含量远高于CD44-/CD24+分化细胞群。敲除内源性TRB3的乳腺癌细胞株,BCSCs的形成能力明显降低；过表达TRB3的乳腺癌细胞株BCSCs的形成和体内成瘤的能力显著升高。说明TRB3促进IL6诱导的BCSCs生成及维持。通过检测TRB3对促干细胞蛋白Sox2、Oct4、Nanog表达及Sox2启动子活性的影响,我们发现TRB3促进Sox2的基因转录。利用siRNA技术筛选调节Sox2的关键转录因子,我们发现转录因子Fox01介导TRB3促进Sox2的基因转录。本研究发现TRB3抑制FoxO1的蛋白酶体降解途径,同时增加Fox01基因转录,最终促进Sox2的基因转录。免疫共沉淀的结果证明TRB3通过与Aktl相互作用,抑制Aktl与FoxO1结合,从而抑制FoxO1的蛋白酶体降解途径。大量FoxO1在细胞核内结合Sox2启动子并促进Sox2基因转录。令我们兴奋的发现是Sox2能与FoxOl启动子结合并促进Fox01基因转录,从而在Sox2与Fox01之间形成正反馈环路,最终促进BCSCs的形成。综上所述,炎症因子IL6增加TRB3的表达,增加的TRB3通过抑制Aktl-FoxO1轴的活性,促进Sox2的基因转录。增加的Sox2也促进Fox01的基因转录,形成正反馈调节环路,进而促进BCSCs的形成。本研究首次证明TRB3促进IL6介导的BCSCs形成及维持；阐明了炎症因子IL6促进BCSCs形成的分子机制。TRB3与Akt1相互作用可能是抑制BCSCs维持、抵抗乳腺癌复发及转移的潜在靶点。目前研究证明,BCL6 (B-cell CLL/lymphoma6)在乳腺癌组织中高表达,并促进乳腺癌细胞的侵袭与转移。但具体的分子机制有待阐明。EMT是肿瘤细胞发生侵袭与转移的关键步骤,在乳腺癌的发生与发展中发挥重要作用。因此我们提出假说,BCL6通过诱导EMT的发生促进乳腺癌的侵袭与转移。利用Transwell、 Anchorage-independent growth和细胞免疫荧光实验,我们发现BCL6通过诱导EMT的发生促进乳腺癌细胞的生长、侵袭与转移能力。BCL6通过抑制上皮细胞标志蛋白E-cadherin的基因转录,促进EMT的发生。利用siRNA技术筛选抑制E-cadherin基因转录的转录因子,我们发现ZEB1介导BCL6诱导EMT和侵袭与转移的能力。利用染色质免疫共沉淀技术,我们发现BCL6促进ZEB1结合E-cadherin启动子,抑制E-cadherin的基因转录。同时我们发现BCL6能与ZEB1启动子结合,促进ZEB1基因转录。说明BCL6通过促进ZEB1基因转录,抑制E-cadherin的基因转录。并且BCL6抑制剂79-6抑制BCL6在乳腺癌细胞中的以上功能。综上所述,BCL6促进ZEB1基因转录,ZEB1对E-cadherin的转录抑制作用增加,最终促进乳腺癌细胞发生EMT。本研究首次证明BCL6促进乳腺癌细胞发生EMT；阐明了BCL6促进乳腺癌发生侵袭与转移的分子机制。BCL6有可能成为抵抗乳腺癌侵袭与转移的潜在靶标。
[Abstract]:Breast cancer is the most common malignant tumor in the world. Although surgery, radiotherapy and chemotherapy-assisted combined therapy for breast cancer have been successful, 40% of patients with breast cancer have a recurrence of tumor and metastasis of cancer cells after treatment, leading to the death of breast cancer patients. Recent studies have shown that breast cancer stem cells (BCSCs) are the key causes of the recurrence, metastasis and resistance of breast cancer. It is proved that Epithelium-Mesenchymal transition (EMT) is a necessary route for the invasion and metastasis of tumor cells. On the other hand, the literature reports that the breast cancer cells of EMT have the characteristics of BCSCs. The purpose of this thesis is to study the mechanism of recurrence and metastasis of breast cancer from two aspects of BCSCs and EMT. A large number of studies have shown that the inflammatory response may be a key cause of the formation and maintenance of BCSCs, but the molecular mechanism of the formation and maintenance of inflammation and BCSCs remains to be clarified. TRB3 (Tribbles homolog 3) is one of the members of the pseudo-protein kinase. Recent studies have shown that TRB3 is not only positively related to the poor prognosis of breast cancer, but also promotes the invasion and metastasis of the tumor. The preliminary work of this lab found that inflammation and stress response increased the expression of TRB3 in the tissue and the tumor tissue of the inflammation. So we put forward the hypothesis that TRB3 mediated the formation and maintenance of BCSCs caused by inflammatory response. The expression of the inflammatory factors IL6, TRB3 and the transcription factor Sox2 in the breast cancer tissue was positively correlated with the detection of the breast cancer tissue chip. Using magnetic bead sorting, we found that the content of TRB3 in the cell group of CD44 +/ CD24-BCSCs induced by IL6 was much higher than that of CD44-/ CD24 + differentiated cells. The formation of BCSCs and the ability of BCSCs in breast cancer cell lines expressing TRB3 were significantly higher than that of BCSCs. Explain that TRB3 facilitates the generation and maintenance of BCSCs induced by IL6. TRB3 was found to promote the gene transcription of Sox2 by detecting the effect of TRB3 on the expression of stem cell protein Sox2, Oct4, Nanog and the activity of Sox2 promoter. The key transcription factor of Sox2 was screened by the siRNA technique, and we found that the transcription factor Fox01 mediates the transcription of the gene of the Sox2 by the transcription factor Fox01. In this study, the proteasome degradation pathway of FoxO1 was inhibited by TRB3, and the transcription of Fox01 gene was increased, and the gene transcription of Sox2 was promoted. The results of the immunoprecipitation demonstrated that TRB3 inhibited the proteasome degradation pathway of FoxO1 by interacting with Aktl to inhibit the binding of Aktl to FoxO1. A large amount of FoxO1 binds to the Sox2 promoter in the nucleus and promotes the transcription of the Sox2 gene. It is found that the Sox2 can be combined with the FoxO1 promoter and promote the transcription of the Fox01 gene, so that a positive feedback loop is formed between the Sox2 and the Fox01, and finally the formation of the BCSCs is promoted. In conclusion, the inflammatory factor IL6 increases the expression of TRB3, and the increased TRB3 promotes the gene transcription of the Sox2 by inhibiting the activity of the Aktl-FoxO1 axis. The increased Sox2 also promotes the gene transcription of Fox01, forming a positive feedback regulation loop, and further promoting the formation of BCSCs. This study first demonstrated that TRB3 promotes the formation and maintenance of BCSCs mediated by IL6, and illustrates the molecular mechanism for promoting the formation of BCSCs by the inflammatory factor IL6. The interaction between TRB3 and Akt1 may be a potential target to inhibit the maintenance of BCSCs and to resist the recurrence and metastasis of breast cancer. The present study has shown that BCL6 (B-cell CLL/ lymphalo6) is highly expressed in breast cancer tissues and promotes the invasion and metastasis of breast cancer cells. but specific molecular mechanisms need to be clarified. EMT is a key step in the invasion and metastasis of tumor cells, and plays an important role in the occurrence and development of breast cancer. Therefore, we put forward the hypothesis that BCL6 promotes the invasion and metastasis of breast cancer by inducing EMT. We found that BCL6 could promote the growth, invasion and transfer of breast cancer cells by induction of EMT. BCL6 promotes the occurrence of EMT by inhibiting the gene transcription of the epithelial cell marker protein E-cadherin. Using siRNA technology to screen the transcription factors that inhibit the transcription of E-cadherin gene, we find that ZEB1 mediates the ability of BCL6 to induce EMT and invasion and metastasis. Using the chromatin immunoprecipitation technique, we found that BCL6 promoted the expression of ZEB1 in the E-cadherin promoter and inhibited the gene transcription of E-cadherin. At the same time, we found that the BCL6 can be combined with the ZEB1 promoter to promote the transcription of the ZEB1 gene. This suggests that BCL6 can inhibit the gene transcription of E-cadherin by promoting the transcription of the ZEB1 gene. and the BCL6 inhibitor 79-6 inhibits the above function of the BCL6 in the breast cancer cell. In conclusion, the BCL6 promotes the transcription of the ZEB1 gene, and the transcription inhibition effect of the ZEB1 on E-cadherin is increased, and finally, the EMT of the breast cancer cell is promoted. In this study, BCL6 was used to promote the development of EMT in breast cancer cells, and the molecular mechanism of BCL6 to promote the invasion and metastasis of breast cancer was explained. BCL6 is a potential target to resist the invasion and metastasis of breast cancer.
【学位授予单位】：北京协和医学院
【学位级别】：博士
【学位授予年份】：2015
【分类号】：R737.9

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