内质网应激相关基因DDIT3调控肿瘤细胞自噬与凋亡的分子机制研究
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摘要
癌症已成为人类最主要的致死疾病之一,严重威胁着人类的寿命。我国的肿瘤发病情况更是不容乐观。根据世界卫生组织发布的《全球癌症报告2014》,2012年全世界共新增1400万癌症病例并有820万人死亡。其中,中国新增307万癌症患者并造成约220万人死亡,分别占全球总量的21.9%和26.8%。肺癌仍然是全世界发病率和致死率最高的癌症,2012年新增肺癌病例180万,死亡人数159万,其中中国因肺癌死亡的的人数超过全世界的1/3。化学疗法是目前治疗癌症的重要手段之一,然而临床上应用的化疗药物还存在着副作用强和引发耐药性等问题,因此寻找安全高效的化疗药物就显得尤为重要。
内质网是真核细胞中蛋白质合成、折叠与分泌的重要细胞器。内源或外源的刺激会导致内质网的蛋白质折叠功能紊乱所引起的一种细胞应激状态称为内质网应激。内质网应激会通过非折叠蛋白反应来清除内质网中的错误折叠蛋白并维持内质网的稳态。如果内质网应激无法逆转,会引起细胞功能恶化进而引起细胞的死亡。越来越多的证据表明,内质网应激在多种肿瘤中广泛存在,并与肿瘤细胞的存活和耐药性密切相关。因此,针对内质网应激信号通路设计和开发抗肿瘤靶向性药物成为目前研究的热点。
细胞自噬和细胞凋亡是调控肿瘤细胞命运的两个重要过程,但二者之间的关系尚不清楚。在肿瘤细胞中,内质网应激既可通过细胞自噬影响细胞的存活或死亡,又可直接诱导细胞死亡。但内质网应激反应如何通过调控肿瘤细胞的自噬和凋亡来决定细胞的命运尚不完全清楚。因此,探讨肿瘤细胞中内质网应激对细胞自噬和细胞凋亡的影响具有重要的理论意义,并且在靶向化疗药物的设计和开发上具有实际的应用价值。
内质网应激是由未折叠蛋白或错误折叠蛋白在内质网腔内积累所引起的。其中未折叠蛋白能使内质网分子伴侣HSPA5和与其结合的三种内质网应激感受器蛋白EIF2AK3、ERN1和ATF6解离,进而该三种跨膜蛋白得以激活。激活后的ERN1通过非常规形式对XBP1前体mRNA的一段26nt的内含子进行剪切,剪切后的mRNA发生翻译框移码,编码成为具有转录活性的转录因子XBP1S (X box binding protein1splicing)。XBP1S作为转录因子可以上调参与内质网相关性蛋白降解途径(ER associated degradation, ERAD)的相关基因的表达,以调节内质网对蛋白质质量的控制和维持氧化还原状态平衡。同时,ERN1还可以招募TRAF2和ASK1基因以激活p38MAPK和.INK MAPK信号通路。EIF2AK3通过介导转录起始因子EIF2A的磷酸化进而抑制蛋白质翻译而使细胞进入暂时的“静止期”。在这种情况下,转录因子ATF4以不依赖帽状结构的方式进行选择性的翻译,上调参与维持内质网稳态的基因的表达。从HSPA5释放后,ATF6转移至高尔基体,由高尔基体蛋白酶SIP (site1protease)和S2P (site2protease)进行切割。被切割的片段作为转录因子调节参与ERAD和维持内质网稳态的几个相关基因的表达。在UPR过程中,DDIT3是UPR的3个感受器共同的靶基因,即ERN1、ATF6和EIF2AK3均可以上调CHOP表达。
DDIT3最早作为一个C/EBPs的成员被鉴定出来,发挥着C/EBPs的显性负抑制剂的作用。DDIT3又被称GADD153、CHOP和C/EBP。C/EBPs作为一个转录因子家族对多种基因进行调控,这些基因广泛参与免疫功能、细胞分化和细胞增殖等生理学过程。目前为止,已经鉴定出6个C/EBP家族成员,分别为：C/EBPα、C/EBPβ、C/EBPγ、C/EBPδ、C/EBPε和DDIT3。DDIT3与C/EBP家族其他成员或JUN/FOS家族成员形成异源二聚体,从而作为转录因子参与在应激过程中诱导表达的基因的转录调控。DDIT3蛋白含有两个功能结构域：一个N端转录激活域(transactivity domain)和一个C端的碱性-亮氨酸拉链(basic leucine zipper, bZIP)结构域。已有的文献证实,DDIT3作为转录因子既可介导BBC3和BCL2L11的转录,又可介导TNFRSF10B的转录从而参与内质网应激诱导的细胞凋亡过程。而最近的研究表明,DDIT3还可以对许多自噬相关基因进行间接或直接调控(如DDIT3作为转录因子通过调节TRIB3和ATG5的表达参与细胞自噬),从而参与由细胞自噬介导的促存活过程。因此,DDIT3可能通过调控细胞凋亡和细胞自噬这两个相互拮抗的生物过程之间的平衡进而影响细胞的命运。然而,DDIT3是否还存在其他诱导细胞自噬的分子机制以及DDIT3诱导的细胞自噬与细胞凋亡的关系等问题尚不清楚。
因此,本研究课题旨在探讨和研究内质网应激相关基因DDIT3如何调控肿瘤细胞的自噬和凋亡；DDIT3所介导的细胞自噬和细胞凋亡之间的关系以及DDIT3介导的细胞自噬和细胞凋亡对肿瘤细胞命运的影响。以期为内质网应激反应对细胞自噬和凋亡的调控机制提供新的实验证据,并为抗肿瘤的靶向化疗药物的设计和开发提供依据。
1.内质网应激相关基因DDIT3调控肿瘤细胞自噬的分子机制研究
1.1DDIT3作为转录因子调控肿瘤细胞自噬的分子机制研究
Salinomycin(盐霉素)是第一个通过高通量的筛选系统鉴定出的能特异性杀死肿瘤干细胞的化合物。该化合物能选择性的杀死乳腺癌及其他肿瘤干细胞,但其具体的分子机制尚不清楚。因而了解其作用机制对于研究肿瘤干细胞特性、肿瘤耐药及开发新型抗肿瘤药物具有重要的意义。在人的非小细胞肺癌细胞中,我们发现：(1)Salinomycin能明显上调MAP1LC3B蛋白的表达；(2) Salinomycin能诱导代表自噬小体的荧光亮点聚集；(3)通过siRNA干扰技术抑制自噬相关基因ATG5和ATG7的表达或利用自噬早期抑制剂(3-MA或LY294002)抑制自噬流后,Salinomycin诱导的MAP1LC3B的蛋白表达明显下调并且荧光亮点明显减少；(4)利用自噬晚期抑制剂(bafilomycin A1或chloroquine)抑制自噬流后,Salinomycin诱导的MAP1LC3B的蛋白表达明显上调并且荧光亮点明显增加。这些结果证实,在人的非小细胞肺癌细胞中,Salinomycin诱导了细胞自噬。我们在进一步的研究中发现Salinomycin诱导的细胞自噬与内质网应激相关：(1)Salinomycin能明显上调内质网应激相关蛋白ERN1、p-EIF2A、ATF4和DDIT3的表达,并能诱导XBP1mRNA的切割；(2)利用siRNA技术抑制ATF4和DDIT3后,Salinomycin诱导的MAP1LC3B蛋白表达明显下调；(3)利用化合物4-PBA抑制内质网应激过程后,Salinomycin诱导的MAP1LC3B蛋白表达明显下调。通过对Salinomycin诱导细胞自噬分子机制进一步的研究,我们证实Salinomycin通过内质网应激抑制MTOR通路,即ATF4-DDIT3/CHOP-TRIB3-AKT1-MTOR通路诱导细胞自噬的发生。此外,我们还发现在人的非小细胞肺癌细胞中,Salinomycin诱导的细胞自噬对促进细胞存活具有重要的作用。另外,为了证实Salinomycin在肿瘤干细胞样的细胞中是否存在相同的机制,我们利用A549细胞构建了能稳定抑制E-cadherin基因CDH1表达的A549/shCDHl细胞系。我们发现Salinomycin在A549/shCDH1细胞中同样能通过ATF4-DDIT3/DDIT3-TRIB3-AKT1-MTOR通路诱导细胞自噬；同时我们还发现A549/shCCDH1细胞对Salinomycin更为敏感,这表明通过抑制细胞自噬对肿瘤干细胞进行靶向治疗可能是最有希望的策略之一。总之,我们的这些发现表明利用Salinomycin联合细胞自噬抑制剂可能是杀死肿瘤细胞和肿瘤干细胞的一种有效的治疗手段。
1.2DDIT3通过与ATG5相结合调控肿瘤细胞自噬的分子机制研究
DDIT3作为转录因子通过调控TRIB3或ATG5的表达间接或直接诱导细胞自噬,但对于DDIT3是否还存在其他诱导自噬的途径尚不清楚。在本课题中,我们发现DDIT3通过三个方面对ATG5进行调节进而调控自噬过程：1)我们在A549、H157、H1792和293FT细胞中发现DDIT3能上调ATG5的表达,与最近的文献报道一致,即DDIT3作为ATG5的转录因子促进ATG5的表达；2)我们通过免疫共沉淀实验证实DDIT3可以与ATG5、ATG12-ATG5复合物及ATG16L1相结合,并且免疫荧光实验结果也显示,在细胞质中DDIT3与ATG5及ATG16L1存在共定位现象；另外,通过GST pull-down实验我们发现DDIT3能促进ATG12-ATG5复合物与ATG16L1的结合以形成ATG12-ATG5-ATG16L1复合体,进而促进自噬小体的形成；(3)根据已有的文献报道,钙网蛋白Calpain可在ATG5的Thr193进行剪切,形成24kD和9kD两个片段,24kD的ATG5片段会转移至线粒体外膜上进而介导细胞凋亡。在本课题中,我们发现DDIT3可与ATG5的C端的区域(194-274aa)结合,并且当DDIT3过表达时可明显抑制由doxorubicin或TG诱导的钙蛋白酶calpain对ATG5的剪切；另外,我们发现DDIT3可以减缓ATG5的降解的降解速度,进而维持ATG5的稳定。此外,我们初步探讨了ATG5也可对DDIT3进行调控：1)我们发现当ATG5过表达时可明显抑制DDIT3的降解速度,进而维持DDIT3的稳定性；(2)我们发现在A549细胞中,TG、cisplatin、doxorubicin及etoposide可诱导ATG5从细胞质转移至细胞核中并增强了DDIT3对TNFRSF10A和TNFRSF10B的反式激活作用。总之,在本课题中我们首次揭示了DDIT3可通过促进ATG5的表达、抑制其降解和促进ATG12-ATG5-ATG16复合体的形成等三个方面参与对自噬的调控,并且我们首次初步探讨了ATG5对DDIT3的调控。这些发现将丰富我们对DDIT3调控自噬和凋亡的分子机制的认识。ATG5和DDIT3之间的相互调控可能是促进细胞自噬与凋亡之间串扰的新机制。
2.内质网应激相关基因DDIT3调控肿瘤细胞凋亡的分子机制研究
2.1DDIT3通过上调TNFRSF10A和TNFRSF10B的表达介导肿瘤细胞凋亡的分子机制研究
肿瘤坏死因子受体超家族成员10a (TNFRSF10A,又称DR4或TRAIL-R1)和肿瘤坏死因子受体超家族成员10b (TNFRSF10B,又称DR5或TRAIL-R2)是两种细胞表面受体,可与肿瘤坏死因子相关的凋亡诱导配体(TRAIL)结合从而介导外源凋亡途径。已有的文献证实,内质网应激诱导剂可通过TNFRSF10A和TNFRSF10B介导细胞凋亡。大量文献证实,DDIT3可以作为转录因子通过调控TNFRSF10B的转录进而上调TNFRSFIOB的表达介导细胞凋亡。然而,对TNFRSF10A的转录调控的分子机制尚不清楚。在本课题中,我们利用内质网应激诱导剂TG和Tm处理人非小细胞肺癌细胞A549、H460和H1792后发现：(1)DDIT3和TNFRSF10A的表达均明显上调,且DD1T3的表达上调时间较TNFRSF10A要早些；(2)DDIT3过表达后TNFRSF10A的表达明显增加；(3)利用siRNA干扰技术抑制DDIT3表达后,TNFRSF10A的表达明显减少。这些结果证实DDIT3可调控TNFRSF10A的表达。为证实DDIT3是否作为转录因子调控TNFRSF10A的转录,我们预测并证实在TNFRSF10A启动子区存在两个假定的DDIT3结合位点(-1636/-1625；-371/-364)和一个假定的AP-1结合位点(-304/-298)。通过萤光素酶活性实验,我们证实在三个假定的结合位点中AP-1结合位点的功能最为活跃。而且,我们发现在TNFRSF10A启动子区的AP-1结合位点(-304/-298)区域,DDIT3可以和phospho-JUN直接结合从而形成DDIT3/phospho-JUN异源二聚体。此外,我们发现一种重要的组蛋白乙酰转移酶KAT2A和DDIT3存在物理性结合。为了探究KAT2A是否也参与TNFRSF10A/B的转录调控,我们进行了3个实验并发现：(1)利用siRNA干扰技术抑制KAT2A的表达后TNFRSF10A/B的表达会明显下调；(2)利用萤光素酶活性检测实验我们发现在KAT2A表达抑制时TNFRSF10A/B启动子的活性明显下调；(3)通过染色质免疫共沉淀(ChIP)实验,我们发现KAT2A可能参与了KAT2A/DDIT3/phospho-JUN复合体或KAT2A/DDIT3复合体的形成,KAT2A可能通过乙酰化H3K9/K14进而促进TNFRSF10A/B的转录。此外,我们还证明了在人的非小细胞肺癌细胞中,TNFRSF10A参与了由内质网应激诱导剂介导的细胞凋亡过程并且呈DDIT3依赖性。总之,我们的研究首次揭示了在内质网应激反应中DDIT3协同乙酰基转移酶KAT2A对TNFRSF10A/B转录调控的分子机制；并且我们还首次证实TNFRSF10A以DDIT3依赖性的方式参与由内质网应激诱导剂介导的细胞凋亡。这些理论成果丰富了我们对细胞凋亡信号通路的认识,同时还为我们设计和开发抗肿瘤化疗药物提供理论依据。
Cancer has become one of the most deadly diseases, which severely threats human beings' life span. The.cancer incidence in our country is even more pessimistic. The world registered14million new cancer cases and8.2million deaths in2012, and the numbers for China were3.07million and2.2million respectively, according to the World Cancer Report2014. China accounted for about21.9percent of the world's new cancer cases in2012and26.8percent of cancer deaths globally. Lung cancer remains the most common and deadliest cancer in the world, with an estimated1.8million new cases and1.59million deaths in2012. More than one-third of such cases occurred in China. The chemotherapy treatment has become an important measure to treat cancer for now, but it can not be denied that the clinically therapeutic drugs exist problems of strong side effects and induce drug-resistance easily. Therefore, to find more safe and effective drugs is evidently important.
The endoplasmic reticulum (ER) is an important organelle for the synthesis, folding and secretion of proteins in eukaryotic cells. Either intrinsic or extrinsic stimulus will lead to the folding dysfunction of ER and thus lead to endoplasmic reticulum stress, one cellular stress state. Endoplasmic reticulum stress can clear away the misfolded proteins in ER. If endoplasmic reticulum stress is not reservible, it will cause cellular malfuntion, which leads to the cell death. More and more evidence has revealed that the endoplasmic reticulum stress exists widely in various tumors, and is closely related to the survival of cancer cells and their resistance to anti-tumor therapy. Thus it is the hotspot of current research to design and develop anti-cancer drugs in view of the endoplasmic reticulum stress signaling pathway.
Autophagy and apoptosis are two important processes to regulate the fate of cancer cells, but the relationship between them remains unclear. In the tumor cells, endoplasmic reticulum stress may influence the survival or apoptosis through autophagy, or induce apoptosis directly. But how endoplasmic reticulum stress regulates autophagy and apoptosis in human cancer cells to determine cellular fates remains largely elusive. Therefore, to study the regulation of endoplasmic reticulum stress to autophagy and apoptosis in tumor cells has its evident theoretical significance and practicable applied value in design and development of targeted chemotherapeutic drugs.
The accumulation of unfolded proteins in the ER cause endoplasmic reticulum stress. These unfolded proteins make the ER molecular chaperon HSPA5disassociate from EIF2AK3, ERN1and ATF6. Upon activation of the ERN1, unconventional splicing initiated by ERN1removes a26-nucleotide intron from unspliced mammalian XBP1mRNA. And the spliced mRNA experiences the translational box frameshift and becomes transcription factor XBP1S (X box binding protein1splicing) with transcription activity. XBP1, as a transcription factor, can up-regulate the expression of genes involved in endoplasmic reticulum associated degredation (ERAD), maintain the quality control of the correct folding of protein in ER and keep the balance of ER in the oxidative and reductive states. In the meanwhile, ERN1can recruit TRAF2and ASK1genes to activate p38and JNK MAPK signaling pathways. EIF2AK3mediates the phosphorylation of transcription initiation factor eIF2a and inhibits protein translation to make cells to enter the temporary'stationary phase1. In this condition, transcription factor ATF4is translated selectively in a cap-independent fashion. ATF4can upregulates the genes involved in the maintainance of ER homeostasis. After released from HSPA5, ATF6translocates to golgi apparatus, where it is spliced by site1protease (SIP) and site2protease (S2P). The spliced fragments, acting as transcription factors, are involved in upregulating the expressions of several genes associated with ERAD and ER homeostasis. In the UPR process, DDIT3(c/EBP homologous protein, CHOP) is the target gene of EIF2AK3, ATF6, and ERN1.
CHOP, which is identified as the first member of C/EBPs (CCAAT/enhancer binding proteins), performs its action as a dominant negative inhibitor. CHOP is also called GADD153(growth arrest-and DNA damaged-inducible gene153), DDIT3(DNA damage inducible transcript3) and C/EBPξ. C/EBPs regulate many genes which are widely involved in immunity, cellular differentiation, etc. There are six members of C/EBP family identified, C/EBPα, C/EBPβ, C/EBPγ, C/EBPε and DDIT3. DDIT3can interacts with one of the C/EBP family members or interacts with one of the JUN/FOS family members to form a heterodimer, to regulate the transcription of genes induced in the stress processes as transcription factor. DDIT3protein is composed of two known functional domains, an N-terminal transcriptional activation domain and a C-terminal basic-leucine zipper (bZIP) domain consisting of a basicamino-acid-rich DNA-binding region followed by a leucine zipper dimerization motif. It has been reported that DDIT3, acting as a transcriptional factor, can mediate the transcription of BBC3, BCL2L11and TNFRSF10B, which are involved in ER stress induced apoptosis. The recent study suggests that DDIT3can also regulate many autophagy associated genes directly or indirectly to induce autopahgy, for example, DDIT3can act as a transcriptional factor and upregulate the expressions of TRIB3and ATG5to mediate autophagy, which plays a prosurvival role. Therefore, DDIT3may affect cells' fate by regulating apoptosis and autophagy, two mutually antagonistic biological processes. However, it is still unclear that whether DDIT3can induce autophagy through other pathways or whether DDIT3plays important roles in regulating the relationship between apoptosis and autophagy.
Above all, this study aims at exploring the mechanisms underlying ER stress related gene DDIT3regulating autophagy and apoptosis, and the relationship between autophagy and apoptosis inducing by DDIT3. We hope that our study can provide new evidence about the regulation mechanisms of ER stress in autophagy and apoptosis, and may provide basis for the design and development of anticancer drug.
1. Molecular mechanisms of autophagy regulated by ER stress related gene DDIT3in human cancer cells
1.1Molecular mechanisms of autophagy regulated by DDIT3acting as a transcription factor in human cancer cells
Salinomycin is perhaps the first promising compound that was discovered through high throughput screening in cancer stem cells. This novel agent can selectively eliminate breast and other cancer stem cells, though the mechanism of action remains unclear. Exploring the molecular mechanisms underlying it will help us to develop new anti-cancer drugs. In this study, we discovered that salinomycin could upregulate the expression of MAP1LC3B and increase the average number of EGFP-MAP1LC3B puncta per cell. In addition, we examined whether salinomycin could induce the autophagic flux in cancer cells. Cotreatment with salinomycin and autophagy inhibitors, such as3-MA and LY294002, or genetic inhibition of autophagy by knocking down either the ATG5or ATG7gene decreased MAP1LC3B-Ⅱ formation and reduced EGFP-MAP1LC3B puncta. In contrast, coincubation with salinomycin and autophagy inhibitors, such as bafilomycin A1or chloroquine, increased MAP1LC3B-II formation and EGFP-MAP1LC3B puncta, indicating that salinomycin induces autophagy in human lung cancer cells. In our study, we demonstrated that salinomycin could upregulate ER stress-related proteins such as phospho-EIF2A, ATF4, DDIT3in both a time-dependent and dose-dependent manner in human NSCLC cells. Furthermore, using RNA interference against ATF4or DDIT3and inhibitor of ER stress (4-PBA) in combination with salinomycin, we confirmed that the EIF2A-ATF4-DDIT3axis was the crucial mediator of salinomycin-induced autophagy. Moreover, our data demonstrate that salinomycin reduces activation of AKT1as well as its downstream substrate MTOR. Taken together, we demonstrated that salinomycin stimulated endoplasmic reticulum stress and mediated autophagy via the ATF4-DDIT3/CHOP-TRIB3-AKT1-MTOR axis. Moreover, we found that the autophagy induced by salinomycin played a prosurvival role in human NSCLC cells and attenuated the apoptotic cascade. To verify whether salinomycin also plays an important role in cancer stem-like cells, we infected A549cells with the shCDHl lentivirus to inhibit CDH1expression, then we treated A549/shCDHl cells with salinomycin for indicated times and detected the expressions of autophagy-related genes and apoptosis-related genes by western blot analysis. The data demonstrated that salinomycin triggered more apoptosis and less autophagy in A549cells in which CDH1expression was inhibited, suggesting that the inhibition of autophagy might represent a promising strategy to target cancer stem cells. In conclusion, these findings provide evidence that combination treatment with salinomycin and pharmacological autophagy inhibitors will be an effective therapeutic strategy for eliminating cancer cells as well as cancer stem cells.
1.2Molecular mechanisms of autophagy regulated by DDIT3via interaction with ATG5in human cancer cells
It has been reported that DDIT3acts as transcription factor and upregulates the expressions of TRIB3and ATG5, both of which can mediate autophagy in human caner cells. However, whether DDIT3also induces autophagy in a novel mechanism remains elusive. In this study, we demonstrated that ATG5is regulated by DDIT3in three distinct mechanisms. Firstly, we confirmed that DDIT3could upregulate the expression of ATG5in A549, H157, H1792and293FT cells, which may verify that DDIT3acts as the transcription factor of ATG5and promotes the expression of ATG5. Secondly, the results of immunoprecipitation assay indicated that DDIT3could interact with ATG5, ATG12-ATG5conjugate and ATG16L1, respectively. In addition, the results of immunofluorescence assay suggested that DDIT3can colocalize with endogenous ATG5and ATG16L1, respectively. Furthermore, we confirmed that DDIT3induces the interaction between ATG12-ATG5conjugate and ATG16L1, which promotes the autophagosome formation. Thirdly, we found that ATG5interacts with DDIT3via its C-terminal region (aa194-274). Moreover, we demonstrated that overexpression of DDIT3could inhibit ATG5from cleavage by Calpain into truncated ATG5(24kDa), which indicates that the interaction between DDIT3and ATG5may protect ATG5from cleavage by Calpain. We also verified that overexpression of DDIT3could slow down the degradation of ATG5, indicating that the interaction between them may maintain the stability of ATG5. In turn, overexpression of ATG5also slowed down the degradation of DDIT3, which suggested that the interaction may promote the stability of DDIT3. In addition, we demonstrated that ATG5could translocate into nucleus after treatment of TG, cisplatin, doxorubicin and etoposide. We also discovered that ATG5played an important roles in DDIT3-dependent transactiviy of TNFRSF10A and TNFRF10B. In sum, our findings indicated that DDIT3could induce autophagy via upregulation of ATG5, or via interaction with ATG5, ATG12-ATG5conjugate and ATG16L to promote autophagosome formation or through sustaining the stability of ATG5. In addition, we also demonstrated that ATG5 could translocate into nucleus and promote DDIT3-dependent transactiviy. These data may enrich our understandings of the mechanisms underlying autophagy and apoptosis induced by DDIT3. The interaction between ATG5and DDIT3may trigger novel crosstalk between autophagy and apoptosis.
2. Molecular mechanisms of apoptosis regulated by ER stress related gene DDIT3in human cancer cells
2.1Molecular mechanisms of apoptosis mediated by DDIT3via upregulation of TNFRSF10Aand TNFRSF10B in human cancer cells
Tumor necrosis factor receptor superfamily member10a (TNFRSF10A; also called DR4or TRAIL-R1) and member10b (TNFRSF10B; also called DR5or TRAIL-R2) are two cell surface receptors which bind to tumor necrosis factor-related apoptosis-inducing ligand (TRAIL) and mediate the extrinsic pathway of apoptosis. It has been reported that TNFRSF10A and TNFRSF10B up-regulation can enhance the apoptosis mediated by ER stress inducers. It has been reported that DDIT3acts as a transcription factor to enhance TNFRSF10B expression and triggers ER stress-induced apoptosis. However, more details underlying the regulation of TNFRSF10B by DDIT3have not been explored. In addition, whether TNFRSF10A, another important death receptor, is also regulated by DDIT3and mediates ER stress-induced apoptosis remains elusive. In this study, we found that DDIT3and TNFRSF10A expressions were upregulated by ER stress inducers, such as TG and Tm, in A549, H1792and H460cells, respectively. In addition, we demonstrated that DDIT3overexpression causes TNFRSF10A induction in the indicated four human lung cancer cell lines, while TNFRSF10A induction was inhibited in DDIT3-konckdown cells after treatment with ER stress inducers, suggesting that TNFRSF10A expression is regulated in a DDIT3-dependent manner. To verify whether DDIT3may act as a transcription factor and induce TNFRSF10A expression through binding to the TNFRSF10A promoter, we predicted and confirmed that there were two putative binding sites located at-1636/-1625and-371/-364in the TNFRSF10A promoter, respectively, and one putative AP-1binding site located in located at-304/-298in TNFRSF10A promoter region, which is the most important among them after our verification. Furthermore, we found that DDIT3interacts directly with phospho-JUN and the DDIT3/phospho-JUN heterodimer binds to the AP-1binding site (-304/-298) within the TNFRSFIOA promoter region. In addition, we confirmed that KAT2A physically interacted with DDIT3. Importantly, knockdown of KAT2A evidently downregulated both the expressions of TNFRSF10A and TNFRSF10B and dramatically decreased luciferase activity of the cells transfected with luciferase reporter plasmid containing AP-1binding site (-304/-298) of the TNFRSF10A promoter and luciferase activity of the cells transfected with luciferase reporter plasmid containing DDIT3binding site (-276/-264) of the TNFRSF10B promoter. Chromatin immunoprecipitation (ChlP) assays showed that KAT2A may participate in KAT2A/DDIT3/phospho-JUN complex or KAT2A/DDIT3complex and acetylate H3K9/K14to further promote the transcription of TNFRSF10A and TNFRSF10B, respectively. Moreover, we verified that KAT2A is recruited by DDIT3at the DDIT3binding site located at-276A264in TNFRSF10B promoter region and mediates H3K9/K14acetylation to further enhance TNFRSF10B transcription. Our findings highlight two novel mechanisms that underlie ER stress-induced TNFRSF10A and TNFRSF10B expression and apoptosis, which will be helpful to elucidate the mechanisms by which anticancer drugs mediates induces apoptosis.
内质网是真核细胞中蛋白质合成、折叠与分泌的重要细胞器。内源或外源的刺激会导致内质网的蛋白质折叠功能紊乱所引起的一种细胞应激状态称为内质网应激。内质网应激会通过非折叠蛋白反应来清除内质网中的错误折叠蛋白并维持内质网的稳态。如果内质网应激无法逆转,会引起细胞功能恶化进而引起细胞的死亡。越来越多的证据表明,内质网应激在多种肿瘤中广泛存在,并与肿瘤细胞的存活和耐药性密切相关。因此,针对内质网应激信号通路设计和开发抗肿瘤靶向性药物成为目前研究的热点。
细胞自噬和细胞凋亡是调控肿瘤细胞命运的两个重要过程,但二者之间的关系尚不清楚。在肿瘤细胞中,内质网应激既可通过细胞自噬影响细胞的存活或死亡,又可直接诱导细胞死亡。但内质网应激反应如何通过调控肿瘤细胞的自噬和凋亡来决定细胞的命运尚不完全清楚。因此,探讨肿瘤细胞中内质网应激对细胞自噬和细胞凋亡的影响具有重要的理论意义,并且在靶向化疗药物的设计和开发上具有实际的应用价值。
内质网应激是由未折叠蛋白或错误折叠蛋白在内质网腔内积累所引起的。其中未折叠蛋白能使内质网分子伴侣HSPA5和与其结合的三种内质网应激感受器蛋白EIF2AK3、ERN1和ATF6解离,进而该三种跨膜蛋白得以激活。激活后的ERN1通过非常规形式对XBP1前体mRNA的一段26nt的内含子进行剪切,剪切后的mRNA发生翻译框移码,编码成为具有转录活性的转录因子XBP1S (X box binding protein1splicing)。XBP1S作为转录因子可以上调参与内质网相关性蛋白降解途径(ER associated degradation, ERAD)的相关基因的表达,以调节内质网对蛋白质质量的控制和维持氧化还原状态平衡。同时,ERN1还可以招募TRAF2和ASK1基因以激活p38MAPK和.INK MAPK信号通路。EIF2AK3通过介导转录起始因子EIF2A的磷酸化进而抑制蛋白质翻译而使细胞进入暂时的“静止期”。在这种情况下,转录因子ATF4以不依赖帽状结构的方式进行选择性的翻译,上调参与维持内质网稳态的基因的表达。从HSPA5释放后,ATF6转移至高尔基体,由高尔基体蛋白酶SIP (site1protease)和S2P (site2protease)进行切割。被切割的片段作为转录因子调节参与ERAD和维持内质网稳态的几个相关基因的表达。在UPR过程中,DDIT3是UPR的3个感受器共同的靶基因,即ERN1、ATF6和EIF2AK3均可以上调CHOP表达。
DDIT3最早作为一个C/EBPs的成员被鉴定出来,发挥着C/EBPs的显性负抑制剂的作用。DDIT3又被称GADD153、CHOP和C/EBP。C/EBPs作为一个转录因子家族对多种基因进行调控,这些基因广泛参与免疫功能、细胞分化和细胞增殖等生理学过程。目前为止,已经鉴定出6个C/EBP家族成员,分别为：C/EBPα、C/EBPβ、C/EBPγ、C/EBPδ、C/EBPε和DDIT3。DDIT3与C/EBP家族其他成员或JUN/FOS家族成员形成异源二聚体,从而作为转录因子参与在应激过程中诱导表达的基因的转录调控。DDIT3蛋白含有两个功能结构域：一个N端转录激活域(transactivity domain)和一个C端的碱性-亮氨酸拉链(basic leucine zipper, bZIP)结构域。已有的文献证实,DDIT3作为转录因子既可介导BBC3和BCL2L11的转录,又可介导TNFRSF10B的转录从而参与内质网应激诱导的细胞凋亡过程。而最近的研究表明,DDIT3还可以对许多自噬相关基因进行间接或直接调控(如DDIT3作为转录因子通过调节TRIB3和ATG5的表达参与细胞自噬),从而参与由细胞自噬介导的促存活过程。因此,DDIT3可能通过调控细胞凋亡和细胞自噬这两个相互拮抗的生物过程之间的平衡进而影响细胞的命运。然而,DDIT3是否还存在其他诱导细胞自噬的分子机制以及DDIT3诱导的细胞自噬与细胞凋亡的关系等问题尚不清楚。
因此,本研究课题旨在探讨和研究内质网应激相关基因DDIT3如何调控肿瘤细胞的自噬和凋亡；DDIT3所介导的细胞自噬和细胞凋亡之间的关系以及DDIT3介导的细胞自噬和细胞凋亡对肿瘤细胞命运的影响。以期为内质网应激反应对细胞自噬和凋亡的调控机制提供新的实验证据,并为抗肿瘤的靶向化疗药物的设计和开发提供依据。
1.内质网应激相关基因DDIT3调控肿瘤细胞自噬的分子机制研究
1.1DDIT3作为转录因子调控肿瘤细胞自噬的分子机制研究
Salinomycin(盐霉素)是第一个通过高通量的筛选系统鉴定出的能特异性杀死肿瘤干细胞的化合物。该化合物能选择性的杀死乳腺癌及其他肿瘤干细胞,但其具体的分子机制尚不清楚。因而了解其作用机制对于研究肿瘤干细胞特性、肿瘤耐药及开发新型抗肿瘤药物具有重要的意义。在人的非小细胞肺癌细胞中,我们发现：(1)Salinomycin能明显上调MAP1LC3B蛋白的表达；(2) Salinomycin能诱导代表自噬小体的荧光亮点聚集；(3)通过siRNA干扰技术抑制自噬相关基因ATG5和ATG7的表达或利用自噬早期抑制剂(3-MA或LY294002)抑制自噬流后,Salinomycin诱导的MAP1LC3B的蛋白表达明显下调并且荧光亮点明显减少；(4)利用自噬晚期抑制剂(bafilomycin A1或chloroquine)抑制自噬流后,Salinomycin诱导的MAP1LC3B的蛋白表达明显上调并且荧光亮点明显增加。这些结果证实,在人的非小细胞肺癌细胞中,Salinomycin诱导了细胞自噬。我们在进一步的研究中发现Salinomycin诱导的细胞自噬与内质网应激相关：(1)Salinomycin能明显上调内质网应激相关蛋白ERN1、p-EIF2A、ATF4和DDIT3的表达,并能诱导XBP1mRNA的切割；(2)利用siRNA技术抑制ATF4和DDIT3后,Salinomycin诱导的MAP1LC3B蛋白表达明显下调；(3)利用化合物4-PBA抑制内质网应激过程后,Salinomycin诱导的MAP1LC3B蛋白表达明显下调。通过对Salinomycin诱导细胞自噬分子机制进一步的研究,我们证实Salinomycin通过内质网应激抑制MTOR通路,即ATF4-DDIT3/CHOP-TRIB3-AKT1-MTOR通路诱导细胞自噬的发生。此外,我们还发现在人的非小细胞肺癌细胞中,Salinomycin诱导的细胞自噬对促进细胞存活具有重要的作用。另外,为了证实Salinomycin在肿瘤干细胞样的细胞中是否存在相同的机制,我们利用A549细胞构建了能稳定抑制E-cadherin基因CDH1表达的A549/shCDHl细胞系。我们发现Salinomycin在A549/shCDH1细胞中同样能通过ATF4-DDIT3/DDIT3-TRIB3-AKT1-MTOR通路诱导细胞自噬；同时我们还发现A549/shCCDH1细胞对Salinomycin更为敏感,这表明通过抑制细胞自噬对肿瘤干细胞进行靶向治疗可能是最有希望的策略之一。总之,我们的这些发现表明利用Salinomycin联合细胞自噬抑制剂可能是杀死肿瘤细胞和肿瘤干细胞的一种有效的治疗手段。
1.2DDIT3通过与ATG5相结合调控肿瘤细胞自噬的分子机制研究
DDIT3作为转录因子通过调控TRIB3或ATG5的表达间接或直接诱导细胞自噬,但对于DDIT3是否还存在其他诱导自噬的途径尚不清楚。在本课题中,我们发现DDIT3通过三个方面对ATG5进行调节进而调控自噬过程：1)我们在A549、H157、H1792和293FT细胞中发现DDIT3能上调ATG5的表达,与最近的文献报道一致,即DDIT3作为ATG5的转录因子促进ATG5的表达；2)我们通过免疫共沉淀实验证实DDIT3可以与ATG5、ATG12-ATG5复合物及ATG16L1相结合,并且免疫荧光实验结果也显示,在细胞质中DDIT3与ATG5及ATG16L1存在共定位现象；另外,通过GST pull-down实验我们发现DDIT3能促进ATG12-ATG5复合物与ATG16L1的结合以形成ATG12-ATG5-ATG16L1复合体,进而促进自噬小体的形成；(3)根据已有的文献报道,钙网蛋白Calpain可在ATG5的Thr193进行剪切,形成24kD和9kD两个片段,24kD的ATG5片段会转移至线粒体外膜上进而介导细胞凋亡。在本课题中,我们发现DDIT3可与ATG5的C端的区域(194-274aa)结合,并且当DDIT3过表达时可明显抑制由doxorubicin或TG诱导的钙蛋白酶calpain对ATG5的剪切；另外,我们发现DDIT3可以减缓ATG5的降解的降解速度,进而维持ATG5的稳定。此外,我们初步探讨了ATG5也可对DDIT3进行调控：1)我们发现当ATG5过表达时可明显抑制DDIT3的降解速度,进而维持DDIT3的稳定性；(2)我们发现在A549细胞中,TG、cisplatin、doxorubicin及etoposide可诱导ATG5从细胞质转移至细胞核中并增强了DDIT3对TNFRSF10A和TNFRSF10B的反式激活作用。总之,在本课题中我们首次揭示了DDIT3可通过促进ATG5的表达、抑制其降解和促进ATG12-ATG5-ATG16复合体的形成等三个方面参与对自噬的调控,并且我们首次初步探讨了ATG5对DDIT3的调控。这些发现将丰富我们对DDIT3调控自噬和凋亡的分子机制的认识。ATG5和DDIT3之间的相互调控可能是促进细胞自噬与凋亡之间串扰的新机制。
2.内质网应激相关基因DDIT3调控肿瘤细胞凋亡的分子机制研究
2.1DDIT3通过上调TNFRSF10A和TNFRSF10B的表达介导肿瘤细胞凋亡的分子机制研究
肿瘤坏死因子受体超家族成员10a (TNFRSF10A,又称DR4或TRAIL-R1)和肿瘤坏死因子受体超家族成员10b (TNFRSF10B,又称DR5或TRAIL-R2)是两种细胞表面受体,可与肿瘤坏死因子相关的凋亡诱导配体(TRAIL)结合从而介导外源凋亡途径。已有的文献证实,内质网应激诱导剂可通过TNFRSF10A和TNFRSF10B介导细胞凋亡。大量文献证实,DDIT3可以作为转录因子通过调控TNFRSF10B的转录进而上调TNFRSFIOB的表达介导细胞凋亡。然而,对TNFRSF10A的转录调控的分子机制尚不清楚。在本课题中,我们利用内质网应激诱导剂TG和Tm处理人非小细胞肺癌细胞A549、H460和H1792后发现：(1)DDIT3和TNFRSF10A的表达均明显上调,且DD1T3的表达上调时间较TNFRSF10A要早些；(2)DDIT3过表达后TNFRSF10A的表达明显增加；(3)利用siRNA干扰技术抑制DDIT3表达后,TNFRSF10A的表达明显减少。这些结果证实DDIT3可调控TNFRSF10A的表达。为证实DDIT3是否作为转录因子调控TNFRSF10A的转录,我们预测并证实在TNFRSF10A启动子区存在两个假定的DDIT3结合位点(-1636/-1625；-371/-364)和一个假定的AP-1结合位点(-304/-298)。通过萤光素酶活性实验,我们证实在三个假定的结合位点中AP-1结合位点的功能最为活跃。而且,我们发现在TNFRSF10A启动子区的AP-1结合位点(-304/-298)区域,DDIT3可以和phospho-JUN直接结合从而形成DDIT3/phospho-JUN异源二聚体。此外,我们发现一种重要的组蛋白乙酰转移酶KAT2A和DDIT3存在物理性结合。为了探究KAT2A是否也参与TNFRSF10A/B的转录调控,我们进行了3个实验并发现：(1)利用siRNA干扰技术抑制KAT2A的表达后TNFRSF10A/B的表达会明显下调；(2)利用萤光素酶活性检测实验我们发现在KAT2A表达抑制时TNFRSF10A/B启动子的活性明显下调；(3)通过染色质免疫共沉淀(ChIP)实验,我们发现KAT2A可能参与了KAT2A/DDIT3/phospho-JUN复合体或KAT2A/DDIT3复合体的形成,KAT2A可能通过乙酰化H3K9/K14进而促进TNFRSF10A/B的转录。此外,我们还证明了在人的非小细胞肺癌细胞中,TNFRSF10A参与了由内质网应激诱导剂介导的细胞凋亡过程并且呈DDIT3依赖性。总之,我们的研究首次揭示了在内质网应激反应中DDIT3协同乙酰基转移酶KAT2A对TNFRSF10A/B转录调控的分子机制；并且我们还首次证实TNFRSF10A以DDIT3依赖性的方式参与由内质网应激诱导剂介导的细胞凋亡。这些理论成果丰富了我们对细胞凋亡信号通路的认识,同时还为我们设计和开发抗肿瘤化疗药物提供理论依据。
Cancer has become one of the most deadly diseases, which severely threats human beings' life span. The.cancer incidence in our country is even more pessimistic. The world registered14million new cancer cases and8.2million deaths in2012, and the numbers for China were3.07million and2.2million respectively, according to the World Cancer Report2014. China accounted for about21.9percent of the world's new cancer cases in2012and26.8percent of cancer deaths globally. Lung cancer remains the most common and deadliest cancer in the world, with an estimated1.8million new cases and1.59million deaths in2012. More than one-third of such cases occurred in China. The chemotherapy treatment has become an important measure to treat cancer for now, but it can not be denied that the clinically therapeutic drugs exist problems of strong side effects and induce drug-resistance easily. Therefore, to find more safe and effective drugs is evidently important.
The endoplasmic reticulum (ER) is an important organelle for the synthesis, folding and secretion of proteins in eukaryotic cells. Either intrinsic or extrinsic stimulus will lead to the folding dysfunction of ER and thus lead to endoplasmic reticulum stress, one cellular stress state. Endoplasmic reticulum stress can clear away the misfolded proteins in ER. If endoplasmic reticulum stress is not reservible, it will cause cellular malfuntion, which leads to the cell death. More and more evidence has revealed that the endoplasmic reticulum stress exists widely in various tumors, and is closely related to the survival of cancer cells and their resistance to anti-tumor therapy. Thus it is the hotspot of current research to design and develop anti-cancer drugs in view of the endoplasmic reticulum stress signaling pathway.
Autophagy and apoptosis are two important processes to regulate the fate of cancer cells, but the relationship between them remains unclear. In the tumor cells, endoplasmic reticulum stress may influence the survival or apoptosis through autophagy, or induce apoptosis directly. But how endoplasmic reticulum stress regulates autophagy and apoptosis in human cancer cells to determine cellular fates remains largely elusive. Therefore, to study the regulation of endoplasmic reticulum stress to autophagy and apoptosis in tumor cells has its evident theoretical significance and practicable applied value in design and development of targeted chemotherapeutic drugs.
The accumulation of unfolded proteins in the ER cause endoplasmic reticulum stress. These unfolded proteins make the ER molecular chaperon HSPA5disassociate from EIF2AK3, ERN1and ATF6. Upon activation of the ERN1, unconventional splicing initiated by ERN1removes a26-nucleotide intron from unspliced mammalian XBP1mRNA. And the spliced mRNA experiences the translational box frameshift and becomes transcription factor XBP1S (X box binding protein1splicing) with transcription activity. XBP1, as a transcription factor, can up-regulate the expression of genes involved in endoplasmic reticulum associated degredation (ERAD), maintain the quality control of the correct folding of protein in ER and keep the balance of ER in the oxidative and reductive states. In the meanwhile, ERN1can recruit TRAF2and ASK1genes to activate p38and JNK MAPK signaling pathways. EIF2AK3mediates the phosphorylation of transcription initiation factor eIF2a and inhibits protein translation to make cells to enter the temporary'stationary phase1. In this condition, transcription factor ATF4is translated selectively in a cap-independent fashion. ATF4can upregulates the genes involved in the maintainance of ER homeostasis. After released from HSPA5, ATF6translocates to golgi apparatus, where it is spliced by site1protease (SIP) and site2protease (S2P). The spliced fragments, acting as transcription factors, are involved in upregulating the expressions of several genes associated with ERAD and ER homeostasis. In the UPR process, DDIT3(c/EBP homologous protein, CHOP) is the target gene of EIF2AK3, ATF6, and ERN1.
CHOP, which is identified as the first member of C/EBPs (CCAAT/enhancer binding proteins), performs its action as a dominant negative inhibitor. CHOP is also called GADD153(growth arrest-and DNA damaged-inducible gene153), DDIT3(DNA damage inducible transcript3) and C/EBPξ. C/EBPs regulate many genes which are widely involved in immunity, cellular differentiation, etc. There are six members of C/EBP family identified, C/EBPα, C/EBPβ, C/EBPγ, C/EBPε and DDIT3. DDIT3can interacts with one of the C/EBP family members or interacts with one of the JUN/FOS family members to form a heterodimer, to regulate the transcription of genes induced in the stress processes as transcription factor. DDIT3protein is composed of two known functional domains, an N-terminal transcriptional activation domain and a C-terminal basic-leucine zipper (bZIP) domain consisting of a basicamino-acid-rich DNA-binding region followed by a leucine zipper dimerization motif. It has been reported that DDIT3, acting as a transcriptional factor, can mediate the transcription of BBC3, BCL2L11and TNFRSF10B, which are involved in ER stress induced apoptosis. The recent study suggests that DDIT3can also regulate many autophagy associated genes directly or indirectly to induce autopahgy, for example, DDIT3can act as a transcriptional factor and upregulate the expressions of TRIB3and ATG5to mediate autophagy, which plays a prosurvival role. Therefore, DDIT3may affect cells' fate by regulating apoptosis and autophagy, two mutually antagonistic biological processes. However, it is still unclear that whether DDIT3can induce autophagy through other pathways or whether DDIT3plays important roles in regulating the relationship between apoptosis and autophagy.
Above all, this study aims at exploring the mechanisms underlying ER stress related gene DDIT3regulating autophagy and apoptosis, and the relationship between autophagy and apoptosis inducing by DDIT3. We hope that our study can provide new evidence about the regulation mechanisms of ER stress in autophagy and apoptosis, and may provide basis for the design and development of anticancer drug.
1. Molecular mechanisms of autophagy regulated by ER stress related gene DDIT3in human cancer cells
1.1Molecular mechanisms of autophagy regulated by DDIT3acting as a transcription factor in human cancer cells
Salinomycin is perhaps the first promising compound that was discovered through high throughput screening in cancer stem cells. This novel agent can selectively eliminate breast and other cancer stem cells, though the mechanism of action remains unclear. Exploring the molecular mechanisms underlying it will help us to develop new anti-cancer drugs. In this study, we discovered that salinomycin could upregulate the expression of MAP1LC3B and increase the average number of EGFP-MAP1LC3B puncta per cell. In addition, we examined whether salinomycin could induce the autophagic flux in cancer cells. Cotreatment with salinomycin and autophagy inhibitors, such as3-MA and LY294002, or genetic inhibition of autophagy by knocking down either the ATG5or ATG7gene decreased MAP1LC3B-Ⅱ formation and reduced EGFP-MAP1LC3B puncta. In contrast, coincubation with salinomycin and autophagy inhibitors, such as bafilomycin A1or chloroquine, increased MAP1LC3B-II formation and EGFP-MAP1LC3B puncta, indicating that salinomycin induces autophagy in human lung cancer cells. In our study, we demonstrated that salinomycin could upregulate ER stress-related proteins such as phospho-EIF2A, ATF4, DDIT3in both a time-dependent and dose-dependent manner in human NSCLC cells. Furthermore, using RNA interference against ATF4or DDIT3and inhibitor of ER stress (4-PBA) in combination with salinomycin, we confirmed that the EIF2A-ATF4-DDIT3axis was the crucial mediator of salinomycin-induced autophagy. Moreover, our data demonstrate that salinomycin reduces activation of AKT1as well as its downstream substrate MTOR. Taken together, we demonstrated that salinomycin stimulated endoplasmic reticulum stress and mediated autophagy via the ATF4-DDIT3/CHOP-TRIB3-AKT1-MTOR axis. Moreover, we found that the autophagy induced by salinomycin played a prosurvival role in human NSCLC cells and attenuated the apoptotic cascade. To verify whether salinomycin also plays an important role in cancer stem-like cells, we infected A549cells with the shCDHl lentivirus to inhibit CDH1expression, then we treated A549/shCDHl cells with salinomycin for indicated times and detected the expressions of autophagy-related genes and apoptosis-related genes by western blot analysis. The data demonstrated that salinomycin triggered more apoptosis and less autophagy in A549cells in which CDH1expression was inhibited, suggesting that the inhibition of autophagy might represent a promising strategy to target cancer stem cells. In conclusion, these findings provide evidence that combination treatment with salinomycin and pharmacological autophagy inhibitors will be an effective therapeutic strategy for eliminating cancer cells as well as cancer stem cells.
1.2Molecular mechanisms of autophagy regulated by DDIT3via interaction with ATG5in human cancer cells
It has been reported that DDIT3acts as transcription factor and upregulates the expressions of TRIB3and ATG5, both of which can mediate autophagy in human caner cells. However, whether DDIT3also induces autophagy in a novel mechanism remains elusive. In this study, we demonstrated that ATG5is regulated by DDIT3in three distinct mechanisms. Firstly, we confirmed that DDIT3could upregulate the expression of ATG5in A549, H157, H1792and293FT cells, which may verify that DDIT3acts as the transcription factor of ATG5and promotes the expression of ATG5. Secondly, the results of immunoprecipitation assay indicated that DDIT3could interact with ATG5, ATG12-ATG5conjugate and ATG16L1, respectively. In addition, the results of immunofluorescence assay suggested that DDIT3can colocalize with endogenous ATG5and ATG16L1, respectively. Furthermore, we confirmed that DDIT3induces the interaction between ATG12-ATG5conjugate and ATG16L1, which promotes the autophagosome formation. Thirdly, we found that ATG5interacts with DDIT3via its C-terminal region (aa194-274). Moreover, we demonstrated that overexpression of DDIT3could inhibit ATG5from cleavage by Calpain into truncated ATG5(24kDa), which indicates that the interaction between DDIT3and ATG5may protect ATG5from cleavage by Calpain. We also verified that overexpression of DDIT3could slow down the degradation of ATG5, indicating that the interaction between them may maintain the stability of ATG5. In turn, overexpression of ATG5also slowed down the degradation of DDIT3, which suggested that the interaction may promote the stability of DDIT3. In addition, we demonstrated that ATG5could translocate into nucleus after treatment of TG, cisplatin, doxorubicin and etoposide. We also discovered that ATG5played an important roles in DDIT3-dependent transactiviy of TNFRSF10A and TNFRF10B. In sum, our findings indicated that DDIT3could induce autophagy via upregulation of ATG5, or via interaction with ATG5, ATG12-ATG5conjugate and ATG16L to promote autophagosome formation or through sustaining the stability of ATG5. In addition, we also demonstrated that ATG5 could translocate into nucleus and promote DDIT3-dependent transactiviy. These data may enrich our understandings of the mechanisms underlying autophagy and apoptosis induced by DDIT3. The interaction between ATG5and DDIT3may trigger novel crosstalk between autophagy and apoptosis.
2. Molecular mechanisms of apoptosis regulated by ER stress related gene DDIT3in human cancer cells
2.1Molecular mechanisms of apoptosis mediated by DDIT3via upregulation of TNFRSF10Aand TNFRSF10B in human cancer cells
Tumor necrosis factor receptor superfamily member10a (TNFRSF10A; also called DR4or TRAIL-R1) and member10b (TNFRSF10B; also called DR5or TRAIL-R2) are two cell surface receptors which bind to tumor necrosis factor-related apoptosis-inducing ligand (TRAIL) and mediate the extrinsic pathway of apoptosis. It has been reported that TNFRSF10A and TNFRSF10B up-regulation can enhance the apoptosis mediated by ER stress inducers. It has been reported that DDIT3acts as a transcription factor to enhance TNFRSF10B expression and triggers ER stress-induced apoptosis. However, more details underlying the regulation of TNFRSF10B by DDIT3have not been explored. In addition, whether TNFRSF10A, another important death receptor, is also regulated by DDIT3and mediates ER stress-induced apoptosis remains elusive. In this study, we found that DDIT3and TNFRSF10A expressions were upregulated by ER stress inducers, such as TG and Tm, in A549, H1792and H460cells, respectively. In addition, we demonstrated that DDIT3overexpression causes TNFRSF10A induction in the indicated four human lung cancer cell lines, while TNFRSF10A induction was inhibited in DDIT3-konckdown cells after treatment with ER stress inducers, suggesting that TNFRSF10A expression is regulated in a DDIT3-dependent manner. To verify whether DDIT3may act as a transcription factor and induce TNFRSF10A expression through binding to the TNFRSF10A promoter, we predicted and confirmed that there were two putative binding sites located at-1636/-1625and-371/-364in the TNFRSF10A promoter, respectively, and one putative AP-1binding site located in located at-304/-298in TNFRSF10A promoter region, which is the most important among them after our verification. Furthermore, we found that DDIT3interacts directly with phospho-JUN and the DDIT3/phospho-JUN heterodimer binds to the AP-1binding site (-304/-298) within the TNFRSFIOA promoter region. In addition, we confirmed that KAT2A physically interacted with DDIT3. Importantly, knockdown of KAT2A evidently downregulated both the expressions of TNFRSF10A and TNFRSF10B and dramatically decreased luciferase activity of the cells transfected with luciferase reporter plasmid containing AP-1binding site (-304/-298) of the TNFRSF10A promoter and luciferase activity of the cells transfected with luciferase reporter plasmid containing DDIT3binding site (-276/-264) of the TNFRSF10B promoter. Chromatin immunoprecipitation (ChlP) assays showed that KAT2A may participate in KAT2A/DDIT3/phospho-JUN complex or KAT2A/DDIT3complex and acetylate H3K9/K14to further promote the transcription of TNFRSF10A and TNFRSF10B, respectively. Moreover, we verified that KAT2A is recruited by DDIT3at the DDIT3binding site located at-276A264in TNFRSF10B promoter region and mediates H3K9/K14acetylation to further enhance TNFRSF10B transcription. Our findings highlight two novel mechanisms that underlie ER stress-induced TNFRSF10A and TNFRSF10B expression and apoptosis, which will be helpful to elucidate the mechanisms by which anticancer drugs mediates induces apoptosis.
引文
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