PADI3通过调控Sirt2表达抑制结肠癌发生的机制研究

Sirt2 plays an important role in tumorigenesis through catalyze a variety of proteins deacetylation. Inhibitors of Sirt2 can significantly suppress many kinds of tumor cells proliferation and were considered as potential antitumor drugs. Inhibition of Srit2 expression and activity are considered to be an important strategy for the development of antitumor drugs. However, toxicity，side effects, poor solubility and low bioavailability severely limited the clinical application of Sirt2 inhibitors. Further study the function of Sirt2 in tumorigenesis, exploring the molecular mechanisms that suppress Sirt2 expression is important for tumor therapy and anti-tumor drug development. We found for the first time that citrulline catalyzing enzyme PADI3 is specifically expressed in colon tissues, can inhibit the proliferation and migration of colon cancer cells in vitro, and inhibit the tumorigenesis and tumor growth of colon cancer in vivo. The genome chip screening revealed that: PADI3 can inhibit Sirt2 expression, but the molecular mechanisms remain unclear. This project plans to explore the molecular mechanism of PADI3 in inhibiting colon cancer tumorigenesis via suppress Sirt2 expression. Deletion mutations, point mutations, co-IP and other molecular techniques were used to determine the key sequences and sites for PADI3 to play a role in tumor suppressing, which will provides new ideas for clinical antitumor drug development.

Sirt2通过去乙酰化调控多种蛋白活性，在肿瘤发生中起重要作用，Sirt2抑制剂能显著抑制多种肿瘤细胞增殖，是潜在的抗肿瘤药物。抑制Srit2表达活性被认为是抗肿瘤药物研发的重要策略。已报道的Sirt2抑制剂由于毒副作用大，溶解度差和生物利用率低等缺陷严重制约了其在临床中的应用。深入研究Sirt2在肿瘤发生中的功能，寻找抑制Sirt2表达的调控机制，对肿瘤治疗及抗肿瘤药物开发有重要意义。本课题组首次发现：瓜氨酸化催化酶PADI3在结肠组织中特异表达，在体外能抑制结肠癌细胞增殖和迁移，体内能抑制结肠癌肿瘤的成瘤和生长，基因组芯片筛查发现：PADI3能抑制Sirt2表达，但具体的调控机制仍不清楚。本项目计划通过缺失突变，点突变，co-IP等关键分子技术，明确PADI3通过调控Sirt2表达抑制结肠癌发生的分子机制，确定PADI3发挥抑癌功能的关键序列和位点，为结肠癌临床药物的开发提供新思路。

结肠癌发病和致死率居高不下，位居恶性肿瘤死亡人数第二位，早期患者治疗后5年生存率大于90%，但对于中晚期患者而言，治疗后5年生存率不足10%。治疗后的转移和复发是最主要致死原因，而肿瘤细胞的恶性增殖及迁移是结直肠癌复发和转移的主要原因，围绕这一问题，本课题组研究发现：瓜氨酸化催化酶PADI3在癌旁组织中高表达，而在结肠癌组织中低表达，进一步研究发现：1）PADI3在体内能显著降低结肠癌肿瘤的生长速度和成瘤率，在体外能通过诱导G1期停滞抑制结肠癌细胞增殖和克隆形成能力；分子机制研究发现，PADI3在细胞质中能通过下调Sirt2表达来抑制Snail表达，抑制AKT磷酸化水平，并促进p21上调表达，结构域分析发现：C-domain能将PADI3定位于结肠癌细胞的胞质中，这对于PADI3发挥抑癌功能是必须的；2）CKS1作为调控细胞中期的关键基因，在结肠癌中高表达，在体外能促进结肠癌细胞的增殖，在体内能促进肿瘤的生长，PADI3能通过抑制Hsp90和CKS1表达发挥其抑癌功能，是Hsp90和CKS1的有效抑制剂，并且Hsp90对PADI3抑制CKS1的表达是必须的；3）CKS1作为重要的癌基因，不仅能促进肿瘤细胞增殖，还能促进通过上调N-cadherin，和Snail，下调E-cadherin的表达来促进结肠癌细胞的迁移，而PADI3过表达可以阻止CKS1对E-cadherin和Snail的表达调控，但对N-cadherin的表达无明显影响，说明PADI3可以通过调节E-cadherin和Snail的表达来挽救CKS1高表达诱导的细胞迁移。本项目围绕PADI3在结肠癌进展中的功能和作用机制展开深入研究，揭示了PADI3通过抑制结肠癌细胞增殖和迁移发挥抑癌功能的分子机制，为结肠癌药物研发和临床治疗提供了新思路和新线索。