去泛素化酶USP9x在稳定肝癌循环肿瘤细胞团中的作用及机制研究

Circulating tumor cells play a vital role in the hepatocarcinoma metastasis and recurrence. Studies have shown that circulating tumor cell clusters have extremely high metastatic potentials. It is considered that the intercellular junctions make a significant contribution during the metastasis of circulating tumor cell clusters. However, the underlying mechanism still need further investigation. In our study, we found the E-cadherin-related adherent junctions directly affect the stability of circulating tumor cell clusters. Furthermore, as an important regulator in ubiquitin-proteasome system, USP9x expression was increased in parallel with the upregulation of E-cadherin. Therefore, we hypothesized that the rapid increase of USP9x after cell cluster detachment resulted in enhancement of intercellular adherent junctions, then increased the stability of tumor cell clusters and promoted their proliferation and anoikis resistance which leading to metastases formation finally. For further investigation, we will explore the function of USP9x and E-cadherin on stabilization of hepatocarcinoma circulating tumor cell clusters and the specific relationship between them basing on an inducible Tet-on expression viral system. Clarification of this phenomenon and its underlying mechanism would enrich current knowledge on metastasis and relapse of HCC and provide new methods and developments on novel targeted drugs for HCC therapy.

研究表明循环肿瘤细胞团转移潜能极高，细胞间稳定连接在其中起关键作用，但目前调节机制不明。肝癌中我们发现钙粘蛋白E-cad相关粘附连接直接影响肿瘤细胞团稳定性；USP9x作为蛋白泛素降解重要调节因子，其与细胞连接关键影响因子E-cad的表达与功能发挥密切相关。因此，我们推测肝癌细胞团脱落后，USP9x表达上调可引起E-cad相关细胞连接形成增多，继而增强细胞团稳定性及其内细胞增殖抗凋亡能力，最终促进转移灶形成。本项目拟利用Tet-on慢病毒技术，在体外模拟血流环境及体内循环中，可控的调节肝癌细胞USP9x及E-cad表达，研究两者在循环肿瘤细胞团稳定性及转移能力中的作用，探讨USP9x在合成、降解、转运方面调节E-cad的具体机制。该现象及机制的阐明，有助于深化对肝癌复发转移机制的认识，为肝癌治疗开发新型靶向药物提供崭新思路。

肝癌是全球范围内最常见的恶性肿瘤之一，其术后高复发转移率是限制临床预后的瓶颈。循环肿瘤细胞的存在是肿瘤复发转移的重要原因，既往研究显示肿瘤细胞团尽管在循环中数量少但其形成转移能力显著高于单个循环肿瘤细胞。我们前期研究表明，肝癌肿瘤细胞团形成肺转移的能力明显高于单个肿瘤细胞，在肝癌肿瘤细胞团形成过程中粘附相关蛋白E-cadherin发挥了重要作用；且初步研究显示USP9x的表达水平及活性与E-cadherin 表达密切相关。在本项目研究中，我们在不同的肝癌细胞系中明确了USP9x的表达水平与肝癌细胞在失巢情况下形成肿瘤细胞团、耐受失巢凋亡及转移能力间的关系。利用可诱导的表达调控系统干预肝癌细胞中USP9x的表达，证实了其表达上调能明显增加肿瘤细胞团的形成，提高其耐受失巢凋亡及转移能力。并进一步阐明了USP9x介导的E-cadherin降解下调是该现象发生的重要机制之一，深化了我们对肝癌循环肿瘤细胞团形成过程及转归的认识；此外，我们还通过肝癌中mircoRNA调控去泛素化酶USP9x水平的相关研究，拓展了其与肝癌细胞凋亡相关机制的认识，有望为开发更加有效的肿瘤治疗策略提供新的思路。