TOPK磷酸化c-Jun促进EGFR-TKIs耐药肺癌细胞生长的分子机制研究

发布时间：2018-01-25 15:14

本文关键词： TOPK 肺癌 EGFR-TKIs耐药 c-Jun AP-1　出处：《第四军医大学》2015年硕士论文　论文类型：学位论文

【摘要】：肺癌是严重威胁人类生命健康的重大疾病,也是全球癌症相关性死亡的首要原因,其中NSCLC约占85%。当前,表皮生长因子受体-酪氨酸激酶抑制剂(EGFR-TKIs)在NSCLC治疗方面获得了巨大的成功,已发展成为肺癌治疗的一线药物。尽管如此,临床中大部分EGFR突变阳性患者经过一段时间的治疗后对EGFR-TKIs产生继发性耐药,因此对EGFR-TKIs获得性耐药机制及其临床应对策略的研究成为靶向治疗领域的又一研究热点。现有研究表明,非小细胞肺癌EGFR-TKIs获得性耐药机制主要表现为细胞绕过EGFR直接通过其下游分子来激活肺癌细胞生长的信号通路。因此,越来越多的研究者开始关注EGFR信号通路的下游分子以及与肺癌生长密切相关的一些蛋白激酶,以克服EGFR-TKIs的耐药问题。TOPK是一种由322个氨基酸构成的丝氨酸-苏氨酸蛋白激酶,首次发现于淋巴因子激活的杀伤性T细胞。以往研究表明,TOPK与有丝分裂期间纺锤体的功能密切相关,TOPK的敲除将导致细胞胞质分裂的推迟。近年来的研究发现,TOPK在多种肿瘤细胞中呈强表达,与肿瘤的发生发展密切相关。TOPK与细胞内多个凋亡相关蛋白的活化和表达有关,参与调控细胞的凋亡。已有研究发现,稳定干涉TOPK表达后,可以对细胞内的一些凋亡相关蛋白以及细胞周期蛋白的表达水平产生不同程度的影响,提示TOPK可能在癌症耐药过程中具有重要的作用。AP-1是一个二聚体的转录因子,包含可以与DNA结合的亮氨酸拉链结构域。Jun(c-Jun,Jun B和Jun D)和Fos(Fos,Fos B,Fra-1,Fra2)亚家族是主要的AP-1蛋白。现有证据表明,AP-1在肿瘤细胞的增殖、转化等方面发挥了重要作用。c-Jun是癌基因,c-Jun活化后可以调节靶基因转录,从而参与细胞的生长、分化和凋亡等多种生理过程。c-Jun的调节与AP-1的转录活性密切相关。但c-Jun已知最主要的调节蛋白是JNK(c-Jun amino-terminal kinase,c-Jun氨基末端激酶)。JNK是丝裂原激活的蛋白激酶超家族成员,JNK接受上游信号后被激活,从而与c-Jun结合并且磷酸化2个丝氨酸/苏氨酸丛,Ser63/73和Thr91/93,进而激活c-Jun增强其转录活性。c-Jun磷酸化后转录活性增强,蛋白的半衰期显著延长。但是JNK只在各种应激状态下被激活,其中最有效的方式是导致DNA损伤的UV刺激。然而在肿瘤正常生长状态下不存在UV刺激,JNK的活化水平较低,那么寻找肺癌细胞中新的能够活化c-Jun进而调节AP-1转录活性的蛋白激酶是肺癌研究的关键。众所周知,肺癌细胞能够大量自分泌EGF,促进肿瘤细胞的生长。肿瘤细胞在EGF刺激下不能活化JNK,但可以活化TOPK,并呈剂量依赖性,这就使得EGFR-TKIs耐药肺癌细胞通过TOPK-c-Jun-AP-1通路促进肿瘤细胞生长成为可能。【目的】①确立TOPK在EGFR-TKIs耐药肺癌细胞生长中的重要作用;②阐明TOPK通过磷酸化c-Jun进而促进AP-1转录活性的具体分子机制。【方法和结果】①首先采用组织芯片技术和western blot方法分别在肺癌患者组织和肺癌细胞系中检测了TOPK的表达情况。结果发现在EGFR-TKIs耐药的肺癌患者和肺癌细胞株中都存在TOPK高表达现象。接下来采用慢病毒干涉技术建立了A549 sh MOCK和A549 sh TOPK稳转细胞系,进一步利用CCK8技术和软琼脂克隆形成实验观察干涉TOPK表达对A549 sh MOCK和A549 sh TOPK细胞生长以及对吉非替尼敏感性的影响。结果发现在EGFR-TKIs耐药的A549细胞中下调TOPK的表达可以明显抑制它的增殖和克隆形成能力,并且显著提高它对于吉非替尼的敏感性。而在吉非替尼敏感的肺癌细胞株H358中提高TOPK的表达后,该细胞对吉非替尼的敏感性下降。②采用western blot方法检测了EGFR-TKIs耐药肺癌细胞A549和敏感肺癌细胞H358中TOPK及相关底物的蛋白表达水平,结果发现,TOPK活化水平与c-Jun磷酸化表达水平呈正相关。进一步采用同源建模预测发现TOPK可以和c-Jun相互结合。③通过哺乳动物双杂交系统证明了TOPK与c-Jun存在相互作用。采用免疫共沉淀方法分别在外源性表达TOPK和c-Jun的HEK293细胞和内源性表达的A549细胞中证实了TOPK与c-Jun存在相互作用。进一步利用体外激酶技术发现TOPK对c-Jun全长蛋白具有磷酸化作用。并且通过点突变分析证明TOPK磷酸化c-Jun的氨基酸位点为Ser63和Ser73。且利用一系列western blot实验证实了TOPK对c-Jun的磷酸化作用。④采用AP-1 luciferase报告质粒技术,观察TOPK磷酸化c-Jun对AP-1转录活性的影响,结果发现TOPK磷酸化c-Jun可以显著提高AP-1的转录活性。并采用real time PCR和western blot技术检测AP-1靶基因CCND1和CDC2 m RNA水平和蛋白水平的表达,结果发现TOPK的表达水平与CCND1和CDC2 m RNA和蛋白的表达水平呈正相关。⑤采用裸鼠移植瘤模型,观察在体内TOPK表达对肺癌细胞A549吉非替尼耐药性的影响,结果发现A549 sh TOPK组肿瘤生长速度明显低于A549 sh MOCK组,并且A549 sh TOPK组对吉非替尼的敏感性显著高于A549 sh MOCK组。并利用western blot方法检测各组移植瘤中TOPK、c-Jun及其磷酸化蛋白的表达水平,结果发现下调TOPK的表达能够明显降低c-Jun的磷酸化水平。【结论】通过本课题的研究,我们发现了TOPK在EGFR-TKIs耐药肺癌细胞生长中发挥了重要作用,并阐明了其作用机制。首先,我们发现TOPK在EGFR-TKIs耐药肺癌细胞中高表达,能促进肺癌细胞的生长,并与肺癌细胞EGFR-TKIs耐药性相关;其次,发现TOPK与c-Jun存在相互作用,TOPK通过磷酸化c-Jun第63、73位丝氨酸而活化c-Jun;最后,我们发现TOPK活化c-Jun可以提高转录因子AP-1的转录活性,进而促进AP-1靶基因CCND1和CDC2的表达水平,从而阐明了TOPK通过磷酸化c-Jun提高AP-1转录活性,促进EGFR-TKIs耐药肺癌细胞生长的具体分子机制,为肺癌的生物学治疗提供了TOPK这一新的靶分子,为肺癌个体化治疗的实施提供了一种新的可能性。
[Abstract]:Lung cancer is a serious threat to the life and health of human beings, is the world's leading cause of cancer-related death, which accounts for about NSCLC 85%. at present, epidermal growth factor receptor tyrosine kinase inhibitor (EGFR-TKIs) has achieved great success in the treatment of NSCLC, the development has become the first-line drug in the treatment of lung cancer. However, the mutation positive patients after a period of time after treatment of EGFR-TKIs secondary resistance to most of the EGFR clinic, therefore become a research hotspot to target treatment in the field of research strategy resistance mechanism and clinical response to EGFR-TKIs. The existing research shows that non small cell lung cancer EGFR-TKIs acquired resistance mechanism mainly for cells to bypass the EGFR directly through the the downstream molecules to activate the signal pathway of lung cancer cell growth. Therefore, more and more researchers begin to pay attention to the EGFR signaling pathway Travel and the growth of some protein kinase molecules closely related with lung cancer, in order to overcome the problem of.TOPK resistance of EGFR-TKIs is composed of a 322 amino acid serine threonine protein kinase, first discovered in lymphokine activated killer T cells. Previous studies showed that TOPK and mitotic spindle during the function is closely related to TOPK the knockout will lead to delayed cytokinesis. Recent studies have found that TOPK was highly expressed in tumor cells, and tumor development is closely related to the activation of.TOPK cells with multiple apoptosis related proteins and expression of apoptosis is involved in the regulation of cells. It has been found that stable expression of TOPK interference that can produce different effects on the cells of some apoptosis related proteins and cell cycle protein expression levels, suggesting that TOPK may have in the process of cancer drug resistance .AP-1 is an important transcription factor two dimers, contains a leucine zipper domain.Jun can be combined with DNA (c-Jun, Jun B and Jun D) and Fos (Fos, Fos, B, Fra-1, Fra2) is a major subfamily of AP-1 protein. The available evidence suggests that AP-1 in the proliferation of tumor cells the transformation has played an important role in.C-Jun is an oncogene, c-Jun activation can regulate the transcription of target genes, which are involved in cell growth, differentiation and apoptosis of a variety of physiological processes, the regulation of.C-Jun and AP-1 transcriptional activity are closely related. But c-Jun is known the main regulatory protein is JNK (c-Jun amino-terminal kinase, c-Jun N terminal kinase).JNK is a member of the superfamily of mitogen activated protein kinase, JNK accept upstream signals after being activated, which combined with c-Jun and phosphorylation of 2 serine / threonine Ser63/73 cluster, and Thr91/93, which can activate c-Jun to enhance its transcription Enhance the transcriptional activity of activated.C-Jun phosphorylation, protein half-life was prolonged. But JNK only in various stress conditions is activated, which is the most effective way to cause DNA damage. However, the stimulation of UV in tumor normal growth does not exist under UV stimulation, the activation of JNK is low, so to find the activation of c-Jun and the new regulation of AP-1 transcriptional activity in lung cancer cells is the key protein kinase in lung cancer research. As everyone knows, a large number of lung cancer cells to autocrine EGF, promote the growth of tumor cells. Tumor cell activation of JNK under the stimulation of EGF can not, but can activate TOPK, in a dose dependent manner, which makes EGFR-TKIs drug resistant lung cancer cells promote tumor cell the growth is possible through the TOPK-c-Jun-AP-1 pathway. [Objective] to establish the important role of TOPK in the growth of EGFR-TKIs drug resistance in lung cancer cells; the elucidation of TOPK phosphorylation by c-Ju N and promote the molecular mechanism of the transcriptional activity of AP-1. [results] the first method and by using tissue chip technique and Western blot method. The expression of TOPK were detected in patients with lung cancer and lung cancer cell lines. The results showed that high expression of TOPK exist in EGFR-TKIs resistant patients with lung cancer and lung cancer cell lines. The next A549 sh MOCK interference technology was established and the A549 sh TOPK cell lines stably transfected by lentivirus, further use of CCK8 technology and soft agar experimental observation on the expression of A549 sh interference TOPK MOCK and A549 sh TOPK on cell growth and the effects of gefitinib sensitivity. The results showed that the expression of TOPK in EGFR-TKIs resistant A549 cells can significantly inhibit the proliferation and clone its formation ability, and improve it to gefitinib sensitivity. In gefitinib sensitive lung cancer To improve the expression of TOPK in H358 cells, the cells to gefitinib decreased imatinib sensitivity. Using Western blot method to detect TOPK EGFR-TKIs drug resistant lung cancer cells A549 and sensitive lung cancer H358 cells and related substrate protein expression level, results showed that the activation of TOPK and phosphorylation of c-Jun expression level was positively related to further. Using homology modeling prediction of interaction between TOPK and c-Jun can be found. Through the mammalian two hybrid system proved that the interaction between TOPK and c-Jun. The HEK293 cells and endogenous CO immunoprecipitation method the expression of TOPK and c-Jun respectively. The expression of exogenous A549 cells confirmed the interaction between TOPK and c-Jun. Further by in vitro kinase technology TOPK has found that phosphorylation of c-Jun full-length protein. And through the point mutation analysis demonstrated that amino acid sites of phosphorylation of TOPK c-Jun Ser63 And Ser73. and blot western by using a series of experiments confirmed that phosphorylation of TOPK c-Jun. The AP-1 luciferase reporter plasmid technology, to observe the effect of TOPK phosphorylation on the transcriptional activity of AP-1 c-Jun, it was found that TOPK phosphorylation of c-Jun can significantly improve the transcriptional activity of AP-1. And the expression of real time PCR and Western blot detection technology AP-1 CCND1 and CDC2 m RNA of target gene and protein level, the expression level of TOPK and CCND1 CDC2 and m RNA and protein expression levels were positively correlated. Applying the nude mice model in vivo, to observe the expression of TOPK on lung cancer cell A549 Kyrgyzstan effect for developed resistance, the results showed that A549 sh TOPK group the tumor growth rate was significantly lower than that of A549 SH and A549 sh MOCK group, TOPK group of gefitinib sensitivity was significantly higher than that of A549 SH and Western MOCK group. Using blot method to detect the transplantation In TOPK, the expression level of c-Jun protein and its phosphorylation, found that down-regulation of TOPK expression significantly decreased the phosphorylation of c-Jun. [Conclusion] through this study, we found that TOPK play an important role in the growth of EGFR-TKIs drug resistant lung cancer cells, and to clarify its mechanism. Firstly, we found that the high expression of TOPK EGFR-TKIs in drug resistant lung cancer cells, can promote the growth of lung cancer cells, and EGFR-TKIs lung cancer cell drug resistance; secondly, found the interaction between TOPK and c-Jun, TOPK by phosphorylation of c-Jun at serine 63,73 and activation of c-Jun; finally, we found that the activation of TOPK c-Jun can enhance the transcription activity of AP-1. And then promote the expression of AP-1 target genes CCND1 and CDC2, which explained the TOPK increased AP-1 transcriptional activity by phosphorylation of c-Jun, promote the growth of lung cancer cells resistant to EGFR-TKIs The specific molecular mechanism provides a new target TOPK for the biological treatment of lung cancer, which provides a new possibility for the implementation of individualized treatment of lung cancer.

【学位授予单位】：第四军医大学
【学位级别】：硕士
【学位授予年份】：2015
【分类号】：R734.2

【相似文献】