NUDCD1通过调控PRKACA苏木化修饰介导结肠癌5-Fu化疗抵抗的机制研究

As a conventional treatment for the patients with colon cancer, the therapeutic efficiency of the adjuvant chemotherapy based on 5-fluorouracil (5-Fu) was diminished by the chemoresistance. Although the NUDCD1 was abnormal up-regulated in colon cancer tissues, the role of this gene in the chemoresistance of colon cancer still remained unknown. Our previous study revealed that NUDCD1 inhibited the apoptosis of colon cancer cells induced by 5-Fu; signal pathway analysis showed that NUDCD1 promoted the phosphorylation of CREB and may cause the resistance to 5-Fu in colon cancer cells. Using PKA kinase activity assay, CO-IP and SUMOylation detection, we found that NUDCD1 might interact with SENP1 which regulated PKA activity via catalyzing the de-SUMOylation of PRKACA. This is the key mechanism of cAMP/PKA/CREB signaling pathway in nucleus. We speculated that NUDCD1 could reduce the nuclear translocation of SENP1 by the complex formation of NUDCD1-SENP1. The loss of nucleus SENP1 delayed the de-SUMOylation of PRKACA, and thus, activated cAMP/PKA/CREB signaling pathway, leading the development of 5-Fu resistance. In current project, we focused on the SUMOylation for PRKACA in 5-Fu resistance and discussed the regulation of NUDCD1 mediated cAMP/PKA/CREB signaling pathway by in vitro and in vivo experiments. This study would make a further exploration on the chemoresistance of colon cancer cell line and therefore provide a new strategy and methodology for the early intervention of colon cancer.

以5-氟尿嘧啶（5-Fu）为基础的结肠癌辅助化疗疗效因化疗抵抗效应而降低。目前，结肠癌中上调的癌基因NUDCD1与化疗抵抗的关系未明。前期结果显示NUDCD1抑制5-Fu诱导的结肠癌细胞凋亡；通路分析表明NUDCD1促进CREB磷酸化是产生5-Fu化疗抵抗的潜在原因。我们通过酶活性实验、CO-IP、苏木化检测等发现NUDCD1与SENP1结合，后者入核使PRKACA去苏木化，是调控PKA活性激活cAMP/PKA/CREB途径的关键。推测机制为NUDCD1与SENP1结合减少后者核转位，抑制SUMO-1修饰的PRKACA去苏木化过程，进而提高PKA活性，促进CREB磷酸化激活下游基因产生5-Fu化疗抵抗。本项目拟以5-Fu化疗抵抗过程中激酶苏木化修饰为切入点，通过体内外实验探讨NUDCD1对cAMP/PKA/CREB途径的调控机制，为进一步阐释结肠癌化疗抵抗机制及早期干预提供新的理论和策略。

研究背景：5-Fu肿瘤化疗抵抗的分子机制缺乏深入研究。NUDCD1在肿瘤进展中发挥重要作用，但在结肠癌化疗耐药中功能尚不明确。.研究内容：本课题研究NUDCD1在调控结肠癌5-Fu化疗耐药中的功能，探讨了NUDCD1-SENP1蛋白复合体调控PRKACA-CREB信号通路的分子机制。.结果及意义：1. NUDCD1对结肠癌5-Fu化疗抵抗的影响。增殖实验结果显示，NUDCD1的下调抑制了结肠癌细胞株的增殖；周期分析实验结果显示NUDCD1下调促进结肠癌细胞发生G1/S期阻滞。加入5-Fu处理后，NUDCD1敲低的结肠癌细胞凋亡显著升高，细胞克隆数目明显减少。体内实验结果显示，NUDCD1敲低组肿瘤细胞形成的瘤体对5-Fu更加敏感。2. NUDCD1激活PKA-CREB信号通路促进结肠癌5-Fu化疗抵抗。信号通路抗体芯片显示NUDCD1调控PKA-CREB信号通路活性并在蛋白水平进行了验证，尤其是PRKACA亚基磷酸化水平降低，CREB蛋白核转位受到抑制。分别下调PRKACA表达及抑制其酶活性，发现结肠癌细胞出现了增殖和转移抑制，以及对5-Fu敏感性增强的表型。3. SUMO化修饰调控PRKACA酶活性及其在结肠癌5-Fu化疗抵抗中的作用。我们证实PRKACA蛋白具有SUMO化修饰的赖氨酸位点。利用点突变技术获得PRKACA突变质粒，通过质粒共转，证实PRKACA的K343位点能够与SUMO1蛋白发生共价连接。细胞功能实验证实，K343位点突变的PRKACA质粒回补并不能完全恢复结肠癌细胞对5-Fu的耐受性，表明该位点的SUMO化修饰具有重要功能。4. NUDCD1-SENP1蛋白复合物介导PRKACA蛋白SUMO化修饰的机制及其功能。通过CO-IP、质谱、激光共聚焦技术证实SENP1和NUDCD1蛋白间存在结合；干扰SENP1的表达后，结肠癌细胞中PRKACA的磷酸化水平明显升高，同时伴随细胞核内SUMO化修饰增强，提示PRKACA的SUMO化修饰会促进其磷酸化水平；发现K343位点的SUMO化修饰可以被SENP1抑制；NUDCD1抑制SENP1蛋白的核转位，干扰NUDCD1可降低细胞核内SUMO化修饰水平。上述结果表明，NUDCD1通过抑制SENP1入核，促进PRKACA 343位赖氨酸的SUMO化，从而上调PRKACA的磷酸化，增强结肠癌5-Fu耐药。