细胞周期关键酶CDK4磷酸化修饰LDHA增强糖酵解促进胆囊癌耐药的机制研究

Refractory chemoresistance is the leading cause of the poor prognosis for gallbladder cancer. Exploration of our findings in the past decade has revealed that decreased ROS levels, higher expression of drug pump proteins (MDR1, MRP1, and ABCG2) and anti-apoptotic proteins (Mcl1 and Bcl2) are the main mechanisms that contribute to chemoresistance in gallbladder cancer. These events are strongly associated with aberrant glycolysis in gallbladder cancer cells. Altered glycolysis has not only emerged as the most active research area for drug resistance, but also provided especially promising targets for novel drug discovery. Our clinical findings also suggested that metabolic tumor burden of biliary tract cancer was associated with patients’ prognosis and chemosensitivity. Our preliminary investigations showed for the first time that CDK4, a key kinase of cell cycle control, reprogrammed glycolysis by promoting LDHA activity and protein stability, thus desensitizing gallbladder cancer cells to cisplatin resistance. Furthermore, S128 in LDHA was firstly identified as the functional phosphorylation site modified by CDK4, which affected LDHA activity and stability. Importantly, we are the first to provide a functional link among cell cycle progression, glucose metabolism and drug resistance. In this study, we aim to reveal and verify the proposed mechanisms underlying LDHA phosphorylation-dependent chemoresistance as followed. CDK4 activation in gallbladder cancer cells is supposed to increase LDHA phosphorylation (S128), leading to enhanced LDHA activity and protein stability, which then promotes glycolysis and inhibits glucose oxidative phosphorylation pathway. This metabolic shift results in accelerated ATP production and decreased ROS level, which increases drug pump protein activity and inhibits oxidative stress response. Decreased intracellular drug retention and anti-apoptotic activity is then found, which eventually cause enhanced drug resistance in gallbladder cancer cells. Taken together, we will also provide basis for whether phosphorylated LDHA would be a novel biomarker for chemosensitivity and a therapeutic target for patients with gallbladder cancer.

胆囊癌细胞对化疗药物不敏感已严重影响其预后。总结本团队近十年研究发现低ROS水平、药泵蛋白（MDR1、MRP1及ABCG2）和抗凋亡蛋白（Mcl-1及Bcl2）高表达是胆囊癌耐药的主要机制，且与肿瘤糖酵解异常密切相关。糖酵解增强是肿瘤耐药和靶向药物筛选的研究热点。临床发现胆道肿瘤糖代谢水平与患者化疗敏感性和预后负相关。预实验首次发现细胞周期关键激酶CDK4能调控糖酵解关键酶LDHA的活性与稳定性，促进胆囊癌耐药，并首次揭示CDK4调控LDHA的关键功能磷酸化位点S128，首次阐明肿瘤细胞周期，糖代谢和耐药的关系。本研究拟阐明CDK4对LDHA精细调控机制，揭示CDK4异常活化→糖酵解水平上升，氧化磷酸化水平下降→ATP生成加快，ROS降低→药泵蛋白活力升高，氧化应激反应下降→化疗药物泵出加快，抗凋亡能力增强→胆囊癌耐药的机制，为LDHA的磷酸化作为胆囊癌化疗敏感性标志物与治疗靶点提供依据。

胆囊癌是胆道系统最常见恶性肿瘤，早期诊断困难，根治切除率低，预后差。化疗是其最常用的辅助治疗手段，但耐药是影响胆囊癌化疗疗效的重要原因，因此研究胆囊癌的耐药机制及干预相关环节提高化疗药物疗效具有重要意义。本研究发现细胞周期蛋白依赖性激酶4（CDK4）对胆囊癌细胞吉西他滨耐药性具有重要调控作用，过表达CDK4将增强吉西他滨的杀伤作用，而敲低CDK4将抑制其杀伤作用。进一步研究发现SUMO特异性蛋白酶3（SENP3）也对胆囊癌细胞吉西他滨耐药性具有调控作用，与CDK4相同，过表达或敲低SENP3都将增强或抑制吉西他滨的杀伤作用。此外，CDK4与SENP3存在蛋白相互作用，且CDK4能调控SENP3的蛋白水平，这一机制可能是通过磷酸化修饰SENP3来实现的。通过本研究，我们发现了新的胆囊癌吉西他滨耐药关键基因，为提高胆囊癌化疗疗效提供了理论依据。