SIRT1-USP29-Twist1轴诱导EMT调控乳腺癌肿瘤干细胞干性、转移及耐药的研究

Triple negative breast cancer (TNBC) is the most malignant breast cancer subtype with a poor prognosis. Tumor metastasis and chemotherapeutic resistance is the primary cause of TNBC-related death. Epithelial-mesenchymal transition (EMT) is the central event to control stemness of breast cancer stem cell，metastasis and chemotherapeutic resistance. We previously demonstrated that CDK4/6-mediated activation of DUB3 is essential to deubiquitinate and stabilize Snail1, a key factor for the initiation of EMT. Furthermore,CDK4/6 inhibitor, palbociclib blocks breast cancer metastasis in TNBC model, without affecting primary tumor growth (Nat Commun 2017,first author). However, how to effectively target Twist1, a key factor for the maintenance of EMT in breast cancer remains unclear. In our preliminary study, we demonstrate that the deubiquitinase, USP29 is essential to deubiquitinate, stabilize Twist1 and therefore, promotes EMT and cell migration. Furthermore, the activity of SIRT1 is essential to maintain Twist1 protein level and cell migration through regulating USP29. Based on these results, our working hypothesis is that SIRT1 dependent activation of USP29 promotes EMT, metastasis, chemo-resistance and stemness of cancer stem cells through stabilizing Twist1. Overall, this study will establish the SIRT1-USP29-Twist1 axis as an important regulatory mechanism of stemness of cancer stem cells, metastasis and chemotherapeutic resistance. Knockdown of SIRT1 or the inhibition of SIRT1 by its specific inhibitor will be utilized to block this axis and the effect on metastasis, chemotherapeutic resistance and stemness of cancer stem cells will be investigated in the proposed study, which will provide a rationale for potential therapeutic interventions in the treatment of triple negative breast cancer.

转移和耐药是三阴性乳腺癌患者主要致死原因，而EMT是决定肿瘤干细胞干性、转移及耐药的关键环节。我们针对EMT起始阶段的重要转录因子Snail1发现了影响其蛋白稳定的CDK4/6-DUB3-Snail1轴（ Nat Commun, 2017,一作）。而EMT维持阶段的关键转录因子Twist1蛋白稳定性的调控机制尚不清楚。我们的预实验通过质谱鉴定出的去泛素化酶USP29可增加Twist1蛋白的稳定性从而促进EMT，而这一过程受去乙酰化酶SIRT1的调控，因此我们提出工作假设：SIRT1-USP29-Twist1轴诱导EMT从而增进乳腺癌肿瘤干细胞干性、转移及耐药。本课题旨在验证这一假设，并分别通过敲减SIRT1和USP29及特异性小分子抑制剂阻断SIRT1的活性阐明SIRT1-USP29-Twist1轴在EMT和上述EMT依赖性恶性表型的作用,为三阴性乳腺癌的精准干预提供新成药性靶点。

转移和耐药是导致恶性肿瘤患者死亡的主要原因，而EMT是决定肿瘤干细胞干性、肿瘤转移、耐药的中枢性病理过程。转录因子Twist1是恶性肿瘤维持EMT的关键蛋白，对于恶性肿瘤中EMT依赖的肿瘤干细胞干性维持、肿瘤转移以及耐药等恶性表型具有重要的作用。因此阐明调控Twist1蛋白稳定性或功能的调控机制，将有利于寻找新的抗癌靶点。而Twist1蛋白稳定性在包括乳腺癌在内的恶性肿瘤中的调控机制尚不清楚。特别是作为一个半衰期极其短暂的转录因子，Twist1难以被直接靶向，迄今为止尚未发现直接靶向Twist1的小分子药物。为了阐明在三阴性乳腺癌中调控Twist1的分子机制，本研究围绕调控Twist1蛋白稳定性的去泛素化酶在 分子、细胞、动物模型以及临床样本等多个层面进行了深入的研究：（1），首先通过质谱分析鉴定出去泛素化酶USP29是Twist1的相互作用蛋白；（2），USP29能够对Twist1进行去泛素化修饰后可增进其蛋白稳定性从而调控肿瘤干细胞干性维持、肿瘤转移以及耐药等恶性表型；（3），蛋白激酶CDK1与USP29相互作用，而CDK1介导的USP29磷酸化修饰能够增加其去泛素化酶活性和Twist1的蛋白水平，进而促进肿瘤干细胞干性维持、肿瘤转移以及耐药等恶性表型,而特异性敲低或抑制CDK1的活性则能显著地抑制上述恶性表型。本研究系统阐明了CDK1-USP29-Twist1轴在EMT依赖性恶性表型（包括肿瘤干细胞干性维持、转移、耐药等）的关键作用，为临床有效治疗三阴性乳腺癌转移、克服肿瘤耐药以及肿瘤干细胞引起的复发找到新的治疗靶点。