ALDH2在微管蛋白抑制剂耐药过程中的作用、分子调控机制及化学干预研究

ALDH family is involved into tumor recurrence, metastasis and drug resistance. Among ALDH family members, cytoplasm ALDHs were considered as the main factor to determine ALDH activity and promote malignancy. However, the role of the ALDH2, a mitochondrial subtype, in tumor biology is still not well known. In our precious study, we found that there was an increasing of ALDH activity and an enhancement of cancer stem cell characteristics in microtubule inhibitor resistant cancer cells as compared to counterpart cells. Interestingly, among the ALDH members, only ALDH2 showed an increasing expression in the drug resistant cells. Importantly, silencing ALDH2 by specific siRNA or blocking ALDH2 by disulfiram, an ALDH inhibitor, could decrease the ALDH activity, reverse drug resistance, and attenuate cancer stem cell characteristics in drug resistant cells, suggesting ALDH2 plays a crucial role in the microtubule inhibitor resistance. Furthermore, our data demonstrated that the epigenetic regulation, including DNA methylation and histone modification, might be contributed to the upregulation of ALDH2 in drug resistant cells. Based on the above results, we plan to: 1) elucidate the regulatory mechanisms of the upregulation of ALDH2 in microtubule inhibitor resistant cancer cells, and disclose the precise role of ALDH2 in the maintenance of cancer cell stemness and its mediated microtubule inhibitor resistance, which will be benefit to comprehensively understand the development and progression of tumor drug resistance；2）explore the therapeutic value of targeting ALDH2 by disulfiram in microtubule inhibitor resistance, which will provide a novel path to reverse tumor drug resistance.

ALDH家族被报道参与肿瘤复发、转移及耐药。以往研究表明细胞浆型ALDH是决定ALDH活性与功能的主要因素，而对线粒体型ALDH2的肿瘤生物学角色尚不清楚。我们前期结果表明，微管蛋白抑制剂（TI）耐药细胞干性特征增强，ALDH活性增加，且ALDH2表达明显增高；沉默ALDH2或靶向抑制后，ALDH活性显著下降、耐药能力减弱、干性特征消失，提示ALDH2在TI耐药过程的关键作用。此外，对其表达调控机制研究发现，DNA甲基化和组蛋白修饰扮演着重要角色。然而，关于它的确切失调机制，在耐药过程中的角色，以及靶向干预的治疗价值还需进一步阐明。为此，本课题拟采用多种技术手段：1）阐明ALDH2在耐药细胞的失调机制，并揭示ALDH2在干性维持及其所介导的TI耐药中的角色特点，以期阐释肿瘤耐药的发生发展过程；2）探索靶向干预ALDH2对TI耐药逆转作用的影响，为靶向逆转肿瘤耐药策略提供新途径。

化疗作为非小细胞肺癌（NSCLC）治疗的主要方式，其耐药现象频现严重影响患者的生存期。探寻化疗耐药的分子机制，发现新的耐药靶点和治疗药物，成为一个亟待解决的难题。本研究从分子、细胞和动物水平，探究乙醛脱氢酶2（ALDH2）在化疗耐药中作用、表观遗传调控机制以及基于调控机制基础上的逆转化疗耐药的策略，旨在为阐明肺癌化疗耐药机制，发现新的耐药逆转策略提供药理学基础。主要研究结果如下：.1. ALDH2在肺癌PTX耐药过程中的作用及表观调控机制.与亲本细胞相比，PTX耐药细胞的干性相关通路富集。生物学确证研究发现PTX耐药细胞干细胞转录因子表达增加，干性生物学特征显著增强，ALDH活性显著增高，且发现在ALDH亚型中以ALDH2表达变化最为明显。进一步研究发现在亲本细胞中过表达ALDH2，PTX敏感性减弱，肿瘤恶性生物学特性增加。在耐药细胞中敲低ALDH2，PTX敏感性增强，肿瘤恶性生物学特性减弱。表达调控机制研究发现核转录因子Y亚基α（NFYA）转录激活ALDH2，而常染色质组蛋白赖氨酸甲基转移酶2（EHMT2）转录抑制ALDH2，抑制EHMT2和同时过表达NFYA对ALDH2的调控具有协同作用。综上所述，ALDH2与肺癌PTX耐药相关，ALDH2表达水平受到转录因子NFYA和表观调控酶EHMT2的协同调控。.2. 靶向干预EHMT2/NFYA/ALDH2信号轴逆转肺癌PTX耐药作用及机制.ALDH2抑制剂异黄酮苷（DZN）或双硫仑（DSF）联用PTX显著增加耐药细胞对PTX的敏感性，抑制NCI-H460/PTX异位荷瘤小鼠肿瘤生长，逆转PTX耐药。表观遗传酶EHMT2的反向调控酶JMJD的抑制剂JIB04下调ALDH2，与PTX联用增加PTX耐药细胞对PTX的敏感性，抑制NCI-H460/PTX异位荷瘤裸鼠肿瘤生长，逆转PTX耐药。.综上所述，研究发现ALDH2在肺癌耐药细胞中表达失调并参与肺癌耐药进程，并首次揭示表观调控酶/转录因子的协同调控机制对ALDH2表达调控的影响。此外，我们还发现小分子化合物可通过多种方式实现对ALDH2表达的调控，逆转肺癌耐药。本研究为阐明肺癌耐药的新机制，并为探索新的逆转策略提供了药理依据。