靶向乙醛脱氢酶ALDH4A1选择性杀伤线粒体自噬缺陷肿瘤细胞及其分子机制

Mitophagy is an important physiological process to maintain the redox homeostasis of cells. Low level of mitophagy is reported to be common in multiple tumors and mitophagy defect plays an important role in tumor progression. It is significant to find the specific target in mitophagy-defective tumors and intervene tumor progression. In our preliminary study, the low expression of ULK1-mediated mitophagy defects promote tumor progression in breast cancer. Through SILAC (Stable Isotope Labeling By Amino Acids In Cell Culture) analysis, we identified redox enzymes ALDH4A1 were upregulated in mitophagy-deficient tumor cells, and knockdown of ALDH4A1 can selectively inhibit the growth of mitophagy-deficient tumor cells. Through kinase screening assay, we found PKC inhibitor can down-regulate ALDH4A1 expression. In this proposal, to clarity the effect of ALDH4A1 in maintaining the survival of mitophagy-deficient tumor cells, we will explore the mechanisms of ALDH4A1 in selectively maintaining redox homeostasis of mitophagy-deficient tumor cells. To clarify the high expression of ALDH4A1 in mitophagy-deficient tumor cells, we also investigate the regulation mechanism of ALDH4A1 phosphorylation, ubiquitination and degradation by mitophagy defects in tumor cells. Mechanically, we will explain the role of PKC inhibitors in selectively killing mitophagy-defective tumor cells by inhibit ALDH4A1 expression. Our research will provide a new target for the treatment of patients with mitophagy defects tumors.

线粒体自噬是维持细胞氧化还原平衡的重要生理过程。研究表明多种肿瘤细胞的线粒体自噬能力低下，且线粒体自噬缺陷促进肿瘤恶性进展。因此寻找线粒体自噬缺陷肿瘤细胞的特异性靶点并实施干预意义重大。我们前期发现ULK1低表达介导的线粒体自噬缺陷促进乳腺癌恶性进展；质谱检测发现线粒体自噬缺陷肿瘤细胞中乙醛脱氢酶ALDH4A1高表达；敲低ALDH4A1可选择性抑制线粒体自噬缺陷肿瘤细胞的生长；激酶筛选显示PKC抑制剂能下调ALDH4A1的表达。本课题拟阐明ALDH4A1调控肿瘤细胞氧化还原平衡的机制，明确其对线粒体自噬缺陷肿瘤细胞生存的维持作用；研究PKC磷酸化ALDH4A1抑制其泛素化降解的机制，明确ALDH4A1在线粒体自噬缺陷肿瘤细胞中高表达的原因；探讨PKC抑制剂通过靶向抑制ALDH4A1表达，选择性杀伤线粒体自噬缺陷肿瘤细胞，从而抑制肿瘤恶性进展，为临床上线粒体自噬缺陷肿瘤患者的治疗提供新靶标。

线粒体自噬缺陷介导的乳腺癌恶性进展是患者治疗失败和死亡的主要原因。本课题重点探讨了如何靶向治疗线粒体自噬缺陷的乳腺癌细胞。① 明确线粒体自噬缺陷促进乳腺癌恶性进展：成功构建线粒体自噬能力不同的乳腺癌细胞模型；发现线粒体自噬缺陷促进乳腺癌侵袭、转移，但不影响肿瘤细胞增殖；发现线粒体自噬缺陷促进乳腺癌骨转移。② 阐明ALDH4A1通过增加谷胱甘肽的合成来维持线粒体自噬缺陷肿瘤细胞氧化还原平衡、生长的作用及其分子机制：利用SILAC技术、质谱分析，结合基因优化法原则，我们发现线粒体自噬缺陷细胞的生存必需基因ALDH4A1；功能学研究发现，ALDH4A1的敲低可选择性抑制线粒体自噬缺陷乳腺癌细胞；发现在线粒体自噬缺陷的肿瘤细胞中，ALDH4A1 通过增加谷氨酸的生成，促进谷胱甘肽的合成，继而清除过度累积的ROS，避免肿瘤细胞因ROS过度累积而发生死亡。③ 阐明了ALDH4A1在线粒体自噬缺陷肿瘤细胞中高表达的分子机制：发现ALDH4A1降解减弱是导致其在线粒体自噬缺陷肿瘤细胞中高表达的原因；发现PKC通过磷酸化ALDH4A1 S289位点来抑制ALDH4A1的泛素化蛋白酶体降解，从而促进ALDH4A1在线粒体自噬缺陷肿瘤细胞中高表达。④ 证实了靶向ALDH4A1选择性杀伤线粒体自噬缺陷肿瘤：发现敲除ALDH4A1显著抑制线粒体自噬缺陷肿瘤细胞的增殖，而对正常细胞没有影响；发现PKC抑制剂通过抑制ALDH4A1来选择性杀伤线粒体自噬缺陷肿瘤细胞。