自噬蛋白LC3作为骨架蛋白介导ERK5磷酸化促进结肠癌细胞获得性5-Fu耐药的机制研究

5-Fu has been used as one of the basal drugs in the first line chemotherapy regimens for colon cancer, which approximately accounts for 50% of the total efficacy in the combined chemotherapy. However, acquired 5-FU chemo-resistance in colon cancer has been increasing prominently, which result in disease progression and death of patients. Thus, it’s crucial important to understand the new mechanism of 5-FU chemo-resistance and develop new therapeutics to overcome the drug resistance. The current study revealed that autophagy protein LC3-II served as protein scaffolds for ERK1/2, and increased phosphorylation level of ERK1/2, by linking to 5-Fu resistance due to hyperphosphorylation of ERK1/2, the interaction between LC3-II and ERK1/2 would be a potential new mechanism for 5-Fu resistance in colon cancer cells. Extracellular signal-regulated kinase 5 (ERK5), is the most recently identified member of the mitogen-activated protein kinase (MAPK) family. Aberrant ERK5 signal transduction has been reported in colorectal cancer, and ERK5 activity upregulation was associated with increase of cancer cells proliferation, invasion and metastasis. Inhibition of ERK5 phosphorylation could amplify 5-Fu mediated cell killing in colon cancer cells through p53 depended mechanism. However, the relationship of autophagy, ERK5 phosphorylation and 5-Fu chemoresistance in colorectal cancer has not been reported yet. In our previous study, it was found increase of ERK5 expression and phosphorylation level in acquired 5-Fu resistant colon cancer cells comparing to the parental cells, meanwhile, we identified increase of autophagosome formation in these resistant cells. Inhibition of autopohagoson formation by 3-MA decreased ERK5 phosphorylation level and re-sensitized resistant cell to 5-Fu. The preliminary colon cancer tissue array displayed positive relationship between LC3 protein level and ERK5 phosphorylation level, which greatly increased in colon cancer tissues compared to the adjacent normal colon mucosal cell. All of above demonstrated the possibility for autophagosome served as scaffold for ERK5 activation through interaction of LC3-II and ERK5, which related to acquired 5-FU resistance in colon cancer. Thus, this project will focus on the molecular mechanism discovery for how LC3-II working as a scaffold protein to promote ERK5 phosphorylation, identification of the interaction motif in ERK5 protein, and evaluation of the effect on ERK5 phosphorylation and 5-Fu resistance by two different autophagy inhibition strategies. Further clinical relevant analysis will reveal the relationship of LC3-II promoted ERK5 phosphorylation and 5-Fu resistance, as well as the disease prognosis. Which could promote the clinical translation of the combination of autophagy inhibition and anti-ERK5 strategy and provide a potential direction to the researches for improving the prognosis of 5-FU resistant colorectal cancer.

5-Fu是结肠癌化疗的基础药物，决定近50%疗效，但其获得性耐药日益突出，故深入研究耐药新机制至关重要。研究显示自噬蛋白LC3作为骨架蛋白介导ERK1/2磷酸化，抑制ERK1/2增加结肠癌细胞5-Fu敏感度。ERK5能促进结肠癌增殖及转移，抑制ERK5也增加5-Fu敏感度，而自噬与ERK5的关系未见报道，抑制自噬流对5-Fu增敏的作用仍有争议。我们发现获得性5-Fu耐药结肠癌细胞中自噬体增多及ERK5磷酸化增高，两者荧光共定位，抑制自噬体形成后ERK5磷酸化下降，细胞对5-Fu再敏感；组织阵列分析显示结肠癌中LC3与ERK5磷酸化正相关，与生存率负相关，提示LC3作为骨架蛋白介导ERK5磷酸化促进5-Fu耐药可能。本课题将阐明LC3作为ERK5骨架蛋白的机制，明确抑制自噬流早、晚期调控ERK5及5-Fu耐药的差异，临床数据分析该机制与结肠癌病人5-Fu耐药的相关性，为临床转化提供实验依据。

结直肠癌是我国最常见的恶性肿瘤之一。5-Fu广泛地应用于结直肠癌术后辅助化疗以及转移性癌的治疗，它是多药联合化疗的基础。然而随着5-Fu的广泛应用，其获得性耐药问题越来越突出。结直肠癌获得性5-Fu耐药是多因素调控的过程。联合用药是目前临床上用于逆转结直肠癌5-Fu耐药的有效策略。理解和发现新的耐药机制更是迫在眉睫。本项目主要通过分子生物学、细胞学、动物实验以及临床资料的统计分析等手段，研究了自噬、ERK5与5-Fu耐药之间的关系和调控机制。我们发现ERK5磷酸化水平增高能通过TFCP2-TS轴影响细胞对5-Fu的敏感度，通过高通量筛选获得了能逆转5-Fu耐药的ERK5抑制剂SC1。同时从5-Fu耐药结直肠癌细胞自噬活跃的特征出发，分析了自噬相关蛋白与ERK5磷酸化之间的关系，发现ERK5在磷酸化过程中与自噬体相结合，其中自噬相关蛋白LC3-II起着关键的蛋白骨架作用。敲除ATG5，ATG7或VPS34-IN1抑制自噬体形成，减少了LC3-II的含量，ERK5失去了磷酸化所需要的场所，因而不能被激活，导致结肠癌细胞TS水平下降，而对5-Fu敏感。通过不同ERK5变异体表达，以及与LC3共沉淀发现与LC3结合的ERK5位点位于MEK5结合区域，其中的FKSVY-V-V-LDL（a.a.130-139）序列是LC3与ERK5蛋白结合必要的结构基础。最终我们通过动物实验验证了抑制自噬体形成和抑制ERK5磷酸化联合逆转5-Fu耐药策略的体内可行性，并从临床标本中验证了LC3与ERK5磷酸化水平的正相关性，以及与5-Fu耐药患者预后的负相关性。本项目的明确了自噬-ERK5-TS表达之间的内在分子调节过程，阐明自噬体通过调控ERK5磷酸化介导结肠癌细胞5-Fu耐药的具体机制，为逆转结直肠癌5-Fu耐药治疗中抗ERK5和抗自噬策略的临床转化提供可信的实验依据。