hTERT非端粒酶逆转录酶活性抑制叉头盒蛋白M1泛素化促胃癌侵袭转移的作用及机制研究

Invasion and metastasis account for the major mortality of gastric cancer. Human telomerase reverse transcriptase (hTERT) plays a crucial role in invasion and metastasis and the current studies mainly focus on its inherent reverse transcriptase activity. However, our previous studies showed that hTERTDN, which lacks the reverse transcriptase activity, could still promote invasion and metastasis of gastric cancer with the mechanism unclear. Besides, we found that hTERTDN promotes gastric cancer invasion and metastasis through inhibiting FOXM1 protein degradation. In addition, the results of mass spectrometry and bioinformatics prediction suggested the interaction regions between hTERT and FOXM1 share the same sites with the interaction regions between E3 ubiquitin ligase E3X and FOXM1. Moreover, Co-IP and confocal microscopy revealed that hTERT could interact with FOXM1 and suppress FOXM1 ubiquitination. Given the results above, we proposed the hypothesis that hTERT may upregulate FOXM1 via antagonizing the binding of E3X to FOXM1 and subsequent FOXM1 ubiquitination, which finally leads to FOXM1 stabilization and enhances gastric cancer invasion and metastasis. For the further investigations, we will use biological technologies such as Co-IP and point mutation to identify the biding sites between hTERT or E3X with FOXM1. Furthermore, we will confirm that hTERT promotes gastric cancer invasion and metastasis through inhibiting the E3X-mediated FOXM1 ubiquitination. Taken together, our study may enrich the mechanism of hTERT-induced gastric cancer metastasis and provide novel scientific evidences for hTERT-targeted anti-cancer drug development.

侵袭转移是胃癌死亡的主要原因，人端粒酶逆转录酶 (hTERT)在其中发挥重要作用，目前hTERT促侵袭转移研究主要聚焦于逆转录酶活性。预实验表明逆转录酶活性缺失的hTERT仍可促胃癌侵袭转移，但机制不清。预实验发现hTERT可非依赖逆转录酶活性抑制FOXM1蛋白降解，促胃癌侵袭转移。质谱及生物信息学预测发现hTERT及E3泛素连接酶E3X均与FOXM1存在共同的潜在结合区，Co-IP及共聚焦证实hTERT可与FOXM1互作且抑制FOXM1泛素化。据此提出“hTERT可能通过竞争E3X结合FOXM1进而抑制FOXM1泛素化降解，上调FOXM1促胃癌侵袭转移”的假说。本项目拟采用Co-IP,点突变等解析hTERT及E3X分别和FOXM1的互作位点，阐明hTERT通过抑制E3X介导的FOXM1泛素化促胃癌侵袭转移的作用，以期丰富hTERT促胃癌侵袭转移机制,为靶向hTERT药物研发提供新依据。

胃癌是常见的消化系统恶性肿瘤之一，其患病率及死亡率均位于恶性肿瘤前列。然而，胃癌发生发展的具体机制仍未完全阐明。人端粒酶逆转录酶（hTERT）是端粒酶的核心亚基，可通过维持肿瘤细胞永生化，抑制凋亡等途径促进肿瘤发生发展。叉头盒蛋白M1（FOXM1）是FOX家族成员之一，可转录激活多种致癌基因，在多种肿瘤发生发展中发挥重要作用。既往研究报道FOXM1亦可通过蛋白-蛋白互作等非转录功能发挥促癌作用。然而hTERT与FOXM1的相互调控关系在胃癌发生发展中的作用尚不明确。本项目研究发现hTERT可非依赖其端粒酶逆转录酶活性抑制FOXM1蛋白泛素化，上调FOXM1蛋白水平。同时，hTERT与FOXM1可共同作用于细胞周期相关的信号通路。hTERT及FOXM1均与胃癌进展及患者不良预后相关，hTERT与FOXM1同时高表达时患者预后最差。其次，FOXM1可非依赖于其经典转录功能上调hTERT蛋白水平。FOXM1通过干扰E3泛素连接酶MKRN1与hTERT的相互作用，阻碍hTERT的泛素化。进一步研究表明，hTERT通过与FOXM1蛋白的DNA结合区域（DBD）互作发挥上述作用。最后，hTERT在FOXM1介导的Wnt/β-catenin通路活化中发挥重要作用，进而促进胃癌细胞的增殖。综上，本研究为hTERT及FOXM1相互作用介导胃癌发生发展提供了新的科学依据。