线粒体靶向小分子IR-34调控HSPA9蛋白诱导肿瘤细胞特异性内质网应激的分子机制

The endoplasmic reticulum stress (ER stress) is an emerging hallmark of tumor biology, and has profound effects on tumorigenesis. It is important to develop tumor-specific ER stress inducing strategies in tumor treatment, especially for the treatment of tumor recurrence, metastasis and drug-resistance. Our previous studies have already demonstrated that the heptamethine cyanine small-molecule IR-34 could induce tumor-specific ER stress by targeting cancer cell mitochondria. But, its mechanisms are not elucidated yet. In this study, we further study the ER stress inducing effects and anti-tumor effects of the IR-34, we explore mechanisms related to the small-molecule targeted mitochondria to induce the tumor-specific ER stress. Using our previous established cancer cell lines, mouse xenografts tumor model and clinical tumor specimens, we will investigate the mechanisms of IR-34 binds to and regulates the HSPA9, and how does HSPA9 participate in the regulation of ER stress by regulating the MAM and endoplasmic reticulum functions; and how does HSPA9 activate the PERK-eIF2α-CHOP downstream signaling pathways of ER stress by regulating IP3R1. We will explore the significance of the newly mechanism of endoplasmic reticulum stress and the relative molecular targets for the clinical tumor treatment and prognosis. The successful implementation of this project and the results from this study will strongly help to deepen the understanding of the mechanism of ER stress inducing by targeting the mitochondria, and provide new ideas for the development of tumor-targeted treatment strategies.

内质网应激在肿瘤中起重要作用，发展肿瘤细胞特异性内质网应激策略对肿瘤治疗，尤其对肿瘤复发、转移和耐药具有明显优势。我们前期合成和鉴定了一种小分子IR-34通过靶向调控线粒体实现肿瘤细胞特异性内质网应激策略，对肿瘤靶向治疗效果显著，但具体机制尚不清楚。据此，本项目拟进一步明确IR-34促内质网应激和抗肿瘤效应，以分子靶标HSPA9为重点和突破口，围绕“线粒体靶向小分子调控肿瘤细胞特异性内质网应激的分子机制”这一关键科学问题，研究IR-34如何参与对HSPA9蛋白调控作用；HSPA9如何调控下游分子VADC1影响MAM及内质网功能稳态；HSPA9如何通过调控IP3R1激活下游PERK-eIF2α-CHOP内质网应激信号通路，并探讨内质网应激新机制及相关分子靶标在肿瘤治疗中的意义。该项目的顺利实施有助于加深对基于线粒体为靶点的内质网应激调控机制的认识，也为制定更有效的肿瘤治疗策略提供新思路。

肿瘤细胞特异性内质网应激调控策略在抑制肿瘤复发、转移和耐药等方面具有明显优势。本课题基于前期研究发现了小分子化合物IR-34可通过靶向调控线粒体实现肿瘤细胞特异性内质网应激调控的研究基础，进一步通过合成小分子连接生物素的方法分离潜在靶蛋白，利用蛋白组学、生物信息学、细胞生物学、分子生物学等技术方法，对IR-34如何通过调控线粒体蛋白HSPA9参与对肿瘤细胞特异性内质网应激调控分子机制进行了初步研究。首先，我们进一步研究了IR-34对肿瘤细胞线粒体的靶向作用以及通过诱导内质网应激反应在肿瘤治疗中的重要优势；其次，通过合成连接生物素的小分子研究工具进行蛋白质组学分析、建立HSPA9稳定低表达细胞质等方法研究IR-34通过靶向HSPA9参与对线粒体功能调控和诱导内质网应激反应而发挥肿瘤杀伤作用；然后，利用分子生物学技术研究IR-34通过靶向抑制HSPA9，干预肿瘤细胞MAM以及内质网钙离子稳态，促进钙离子介导的PERK内质网应激信号通路的分子机制；最后，通过阐明MAM及重要调控分子HSPA9在肿瘤细胞中的特异性表达情况，进一步说明基于小分子IR-34揭示的相关内质网应激分子机制在肿瘤治疗中优势。综上所述，本项目在前期研究发现基础上，进一步揭示了基于线粒体蛋白HSPA9为靶标的内质网应激调控新机制，阐明了基于肿瘤细胞MAM关键靶点的干预策略在肿瘤治疗的中重要潜力，为肿瘤治疗提供新的理论支撑和潜在分子靶标，具有重要理论意义和潜在临床价值。项目研究取得的新发现也为后续开展研究奠定基础。