RIP1在三阴乳腺癌中对TRAIL受体介导的细胞自噬调控机制研究

Triple-negative breast cancer (TNBC) is a subtype of breast cancer that is clinically negative for expression of estrogen and progesterone receptors (ER/PR) and HER-2 protein. It is characterized by its unique molecular profile, highly aggressive behavior, distinct patterns of metastasis, and lack of targeted therapies. Tumor necrosis factor-related apoptosis-inducing ligand (TRAIL) is an attractive candidate for cancer therapy because it triggers the extrinsic and/or intrinsic pathway of apoptosis in a variety of tumor cells by engaging the death receptors DR4 and DR5, while sparing most normal cells. However, recent findings show that death receptor can also transmit alternative necroptotic or autopahgy signaling, depending on the cell type and stimulus. Moreover, receptor interacting protein 1 (RIP1) plays an important role in regulation of different types of cell death-mediated by death receptor activation. In our previous study we found that TRAIL can initiate protective autophagy in TNBC cells. Blocking autophagy can induce the selective protein degradation of RIP1 and increase tumor cell sensitivity to TRAIL-induced apoptosis. Furthermore, down-regulating the expression of RIP1 can also enhance the tumuricidal activity of TRAIL against TNBC cells. These results suggest that RIP1 involve in the TRAIL-receptor-mediated protective autophagy in triple negative breast cancer. However, the underlying mechanism is poorly understood. This study will investigate the function of RIP1 in protective autophagy by using genomics and proteomics methods, indentifying the RIP1-interacting protein, relative signaling and regulatory networks. The aim of this study is to reveal the precise molecular mechanism of RIP1 in regulation of death receptor-mediated autophagy, and disclose a cross-talk between autophagy pathway and the ubiquitin - proteasome pathway .Our study may provide new insight into protective autophagy and subsequently allows development of a novel targeted therapy strategies for triple negative breast cancer.

三阴乳腺癌是近几年提出的一类特殊乳腺癌亚型，与其它类型乳腺癌相比具有侵袭性高、易转移复发等特点，目前仍缺乏有效的分子靶向治疗手段。申请者前期研究发现，肿瘤坏死因子相关凋亡诱导配体（TRAIL）能激活三阴乳腺癌细胞内保护性自噬。阻断自噬能增强TRAIL的肿瘤杀伤效应，同时特异性诱导受体相互作用蛋白1（RIP1）降解。不仅如此，直接下调RIP1同样能提高三阴乳腺癌细胞对TRAIL诱导凋亡的敏感性，但其具体分子机制尚未明确。基于以上背景，申请者首次提出RIP1参与调控TRAIL受体介导的保护性自噬，并将利用基因组学及蛋白质组学方法展开研究。以RIP1 相互作用蛋白及其调控网络作为研究切入点，揭示RIP1 作为调节蛋白在保护性自噬中的作用、阐明细胞自噬与泛素-蛋白酶体途径之间的调控机制。在完善自噬理论基础上为三阴乳腺癌靶向治疗提供新策略。

三阴乳腺癌是一类具有高侵袭性、易复发转移的特殊类型乳腺癌，由于缺乏激素受体及HER-2表达，因此缺乏有效的分子靶向治疗手段。申请者研究发现TRAIL能激活三阴乳腺癌细胞内保护性自噬，阻断自噬能增强TRAIL的肿瘤杀伤效应，同时特异性诱导受体相互作用蛋白1（RIP1）降解。不仅如此，直接下调RIP1同样能提高三阴乳腺癌细胞对TRAIL诱导凋亡的敏感性。进一步研究发现，当TRAIL激活死亡受体组成死亡诱导信号复合物时， RIP1蛋白与FADD、TRADD形成riposome的复合物。在阻断RIP1介导的自噬信号通路后，Caspase 8的活性得到增强，进而将RIP1作为底物进行水解，并在抑制保护性自噬信号通路的同时，使凋亡信号通路得以加强。申请人利用基因芯片技术检测发现TRAF2、CARD9 （caspase recruitment domain-containing protein 9）可能参与调控RIP1蛋白的活性，激活NF-кB信号通路，但相关工作仍在深入研究并探索中。与此同时，研究者发现利用干扰有丝分裂药物同样能增强DR5特异性激动性抗体诱导的细胞凋亡。其具体机制是由于JNK的持续激活从而是Bim蛋白发生磷酸化，从而增强了内源性线粒体凋亡途径。不仅如此，上述过程中还伴随着NF-κB依赖性miR-146a的表达，后者进一步抑制乳腺癌细胞的侵袭转移。鉴于多靶点联合治疗肿瘤是未来发展的趋势。研究者在专注本研究的同时，将兴趣点延伸至药物研发相关领域。鉴于国内外关于TRAIL偶联脂质体或PEG的药物在临床前研究获得肯定，结合本课题证明阻断自噬能够增强TRAIL的肿瘤杀伤活性，因此研发以脂质体作为药物载体，以TRAIL联合包括RIP1在内的多靶点治疗药物具有一定可行性。研究才与脂质体药物载体平台的开发，首先尝试以原癌基因Bmi1作为研究切入点和治疗靶点，在联合化疗药物的基础上探讨构建多靶点治疗药物的可行性，取得了令人鼓舞的结果。这为下一步构建“TRAIL/RIP1 inhibitor-liposome”奠定的夯实的基础。本研究在完善自噬理论基础上为三阴乳腺癌靶向治疗提供新思路。