FoxO3对Nrf2转录活性的调控及其在胆管癌耐药中的分子机制

Cholangiocarcinomas (CCCs) are malignancies of the bile duct system that may originate in the liver and extrahepatic bile ducts. Patients with CCCs are often diagnosed at advanced stage. One of the main reasons for its low survival rate is that CCCs exhibits extensive local invasion and frequent regional lymph node metastasis. There is no effective chemotherapeutic treatment due to drug resistance. Little is known about the molecular mechanism underlying chemoresistance in CCCs. Activation of the PI3K/AKT signaling pathway is frequently found in CCCs cells. Enhanced ROS detoxification due to Nrf2 activation has been shown to promote drug resistance in CCCs. We found decreased mRNA and protein levels of Keap1 in FoxO3-depleting cells. Knockdown FoxO3 also led to up-regulation of Nrf2 protein levels, as well as its transcriptional activity. Strikingly, FoxO3 depletion promoted CCCs cell survival upon cisplatin treatment. Interestingly, Keap1 formed protein complex with FoxO3 and can promote the transcription activity of FoxO3. The goal of this project is to characterize the molecular mechanism underlying FoxO3-dependent activation of Nrf2 signaling and its role in ROS homeostasis and chemoresistance in CCCs. We will also elucidate the mechanism as how Keap1 feed back to FoxO3 via protein-protein interactions. Our study will unravel a previously unappreciated role of FoxO3 in drug resistance via crosstalk with Keap1-Nrf2 pathway. The possibility of FoxO3/Keap1/Nrf2 serving as markers predicting chemoresistance in CCCs will be evaluated in clinical samples. Our findings will shed insight into the molecular mechanism underlying the regulation of ROS homeostasis and drug resistance in CCCs.

胆管癌是一种恶性程度极高且预后差的肿瘤。胆管癌对化疗不敏感，耐药是造成化疗失败的最主要原因。胆管癌产生耐药的分子机制尚不明确。我们发现FoxO3失活导致Keap1 mRNA和蛋白水平下调，Nrf2蛋白水平和转录活性增高，胆管癌细胞对化疗药物的敏感性降低；同时还发现Keap1与FoxO3存在蛋白相互作用，并且Keap1能够促进FoxO3的转录活性。目前关于FoxO3与Keap1-Nrf2通路的相互应答以及FoxO3在胆管癌耐药中的分子机制尚无报道。本项目拟研究FoxO3调控Keap1-Nrf2通路的分子机制；阐明FoxO3-Nrf2的互话在调控ROS水平和胆管癌耐药中的生物学功能；探讨FoxO3/Keap1/Nrf2做为预测胆管癌化疗和预后的分子标志物的可行性；揭示Keap1促进FoxO3转录活性的分子机理及其在氧化应激条件下调控细胞内ROS稳态中的地位，从而为治疗胆管癌提供重要的理论依据。

许多人类肿瘤中存在PI3K/AKT过度激活，然而该通路的异常导致肿瘤形成的分子机理仍未充分阐明。FoxOs是受PI3K/AKT负向调控的转录因子，与肿瘤形成密切相关，但对于FoxOs调控肿瘤生长的机制尚不十分清楚。胆管癌是一类起源于胆道上皮细胞的恶性肿瘤，胆管癌患者对化疗敏感性差，但耐药机理尚未充分阐明。Nrf2的激活与胆管癌耐药性及病人不良预后密切相关。高度激活的PI3K/AKT能够激活Nrf2信号通路，但其中具体的分子机制尚不清楚。Keap1是连接Nrf2与Cul3的衔接蛋白，介导Nrf2通过蛋白酶体途径进行降解，目前关于Keap1的转录调控几乎未知。我们研究证实，Keap1是FoxO3的下游靶基因，敲低FoxO3导致 Keap1的转录下调，从而激活Nrf2信号通路。我们进一步研究发现，在胆管癌细胞中持续激活AKT信号或者TNFα处理胆管癌细胞，均能明显抑制FoxO3的表达水平和转录活性，进而抑制Keap1的表达，最终导致Nrf2信号通路的激活。前期报道认为FoxO3通过转录下调MnSOD及catalase可以增强细胞内活性氧水平，但是我们研究发现：缺失FoxO3的细胞通过激活Nrf2可以降低胞内活性氧的水平，从而保护细胞免受氧化压力的伤害。最重要的是，利用裸鼠皮下移植瘤模型，我们发现FoxO3缺失通过激活Nrf2信号通路能够促进胆管癌细胞的体内成瘤能力，同时增强肿瘤细胞对cisplatin的耐药性。在临床胆管癌样本中，我们检测到磷酸化的AKT与Keap1的mRNA水平呈负相关，磷酸化的AKT与Nrf2蛋白水平呈正相关，FoxO3 mRNA与Keap1 mRNA的表达水平呈正相关性，FoxO3 mRNA与Nrf2蛋白的表达水平呈负相关性，这些蛋白的相关性进一步阐明了FoxO3对Keap1-Nrf2信号通路的调控及其在胆管癌肿瘤生长及耐药中的分子机制。

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