肿瘤细胞中BIT1介导内质网应激诱导细胞凋亡的机制研究

To sense and respond to various disturbances, eukaryotic cells have evolved a group of protective pathways, collectively termed the endoplasmic reticulum (ER) stress response. The initial intent of the ER stress response is to restore normal ER function. If the stress is prolonged, or the adaptive response fails, ER stress can also lead to apoptosis. Increasing evidences support that ER stress-associated apoptosis contributes to the pathology of several important human diseases, including cancer. Thus, the induction of ER stress-mediated apoptosis in cancer cells represents an alternative approach in cancer therapy. However, the molecular mechanisms linking ER stress to apoptosis still need to be further demonstrated. Recently, we demonstrated that Bcl-2 inhibitor of transcription (BIT1), an anoikis effector, localized in the secretory pathway, especially in the Golgi complex. Forced localization of BIT1 in the ER not only perturbed the translocation process of endogenous BIT1 to Golgi, also led to enhanced ERK signalling from the Golgi complex. Considering that ER stress directly disturbs the secretory pathway and affects MAPK activation, we put forward the possibility of regulation of BIT1 function by ER stress. Actually, either targeted expression of BIT1 in the ER or silencing of BIT1 expression resulted in enhanced cell resistance to ER stress-induced apoptosis. It is suggested that BIT1 also play a role in ER stress-mediated apoptosis. Firstly,this proposal will confirm the activity of BIT1 in tumor cells apoptosis induced by ER stress. Further we plan to reveal the regulation mechanism of ER stress on BIT1 function, and the mechanism underlying BIT1-mediated apoptosis induced by ER stress by different methods, such as mammalian two-hybrid technology and FRET. This study is expected to throw light on molecular mechanisms of ER stress-induced apoptosis, and provide a new theory for complete demonstration of tumorigenesis and tumor progression.

内质网应激反应是细胞应对多种胁迫的自我保护反应。若刺激过于强烈或持续时间过长，超过了细胞自身的代偿能力，内质网应激亦可诱导细胞凋亡。内质网应激与肿瘤发生发展密切相关，促进内质网应激诱导的凋亡代表着一种新的肿瘤治疗策略，但相关分子机制有待完善。我们研究发现，内质网定向表达失巢凋亡效应分子BIT1或下调其表达可导致ERK通路的活化，同时显著提高细胞对内质网应激诱导凋亡的抗性。由此推测，BIT1可能参与内质网应激决定细胞命运的过程，具体作用机制亟待阐明。申请者计划首先确证BIT1与内质网应激促肿瘤细胞凋亡过程的相关性，进而利用哺乳动物细胞双杂交技术和FRET等方法揭示肿瘤细胞中内质网应激调控BIT1功能的机制，阐明涉及其中的具体分子事件，系统诠释BIT1介导内质网应激诱导细胞凋亡的精细机制，深化当前对内质网应激诱导细胞凋亡机制的认识，为相关病理过程的阐明提供新的理论基础。

内质网中存在一套严格的蛋白质质量控制体系，只允许正确折叠和修饰的蛋白输出至高尔基体。当缺氧、葡萄糖缺乏或钙稳态失衡等造成未折叠或错误蛋白在内质网内大量堆积时，会引发内质网应激。细胞通过启动内质网应激反应或未折叠蛋白反应来恢复内质网稳态。内质网应激反应是一把双刃剑，与细胞的生死命运密切相关。内质网应激反应决定细胞生死命运的转换机制并不清楚。有报道证实，蛋白激酶D（PKD）与氧化胁迫和缺血性损伤条件下的细胞存活密切相关，两种因素均可引发内质网应激。在增殖快和预后差的肿瘤中，PKD的表达显著增加。本研究中，我们首次证实内质网应激能够促进肿瘤细胞中PKD的活化，PKC依赖的PKD活化介导了内质网应激促细胞生存的效应。PKD通过影响经典的内质网应激感受器IRE1的稳定性来严格控制IRE1介导的下游信号。抑制PKD的活性导致IRE1的降解，并下调其下游XBP1s的表达及XBP1s靶基因的表达。不仅如此，活化的PKD亦通过促进MKP-1的磷酸化来抑制IRE1/JNK通路。JNK的活化模式被证实是内质网应激决定细胞生死的关键因素之一。内质网应激条件下，活化的PKD促进JNK激活模式的改变，从促细胞凋亡转变为促细胞生存模式。本研究揭示了一条新的由PKD介导的内质网应激促细胞生存的信号途径，有助于深入阐明内质网应激决定细胞命运的分子机制。我们的研究证实，肿瘤细胞能够通过PKD“劫持”经典的应激反应，使其偏向于发出促细胞生存的信号。目前，尚未研发出一种有效的作用于PKD家族的药物。鉴于PKD与肿瘤发生发展密切相关，我们同时分析了两种PKD抑制剂的作用模式，探究了导致两种抑制剂作用效果显著不同的机制，首次构建了PKD的非ATP竞争性抑制剂的结合模式，为后续高效药物研发奠定了基础。