表观遗传蛋白 EZH2 作为消除脉络膜黑色素瘤肿瘤干细胞干预靶点的确认研究

Uveal melanoma is sporadic but the most common intraocular malignancy in adults. The patient benefit of current treatment of UM is limited by the lack of effective chemotherapeutic agents. Traditional treatment of primary lesions is enucleation of the affected eyes. Almost 50% of UM patients develop liver metastases with poor life expectancy. Presence of cancer stem cells (CSCs) is one of the most important causes of intrinsic resistant to chemotherapeutic agents and liver metastasis. Preliminary rResults from our group showed that epigenetic regulator EZH2 may play a key role in regulating and maintaining self-renewal of CSCs in UM. This study aims to identify EZH2 as a key regulator of CSCs in UM and further clarify it’s a therapeutic target. We will systemically analyze the effect of EZH2 on the expression of cancer stem cell markers and the self-renewal and frequency of CSCs of UM; the regulation effect of EZH2 on pathways involved in CSCs; the role of EZH2 in regulating the migration and invasion of UM cells; further, the apoptosis mechanism induced by EZH2 specific inhibitor. The project may validate EZH2 as the target protein for UM and opening the way for a novel target therapeutic strategy for eliminating UM CSCs.

脉络膜黑色素瘤 (uveal melanoma, UM) 属于罕见而重要的眼科疾病。因缺乏有效化学治疗药物而通常采用摘眼球手术。不管是否手术，约50%病人最终发生肝转移，死亡率100%，肝转移的机制还不清楚。肿瘤干细胞 （cancer stem cells, CSCs）是引起 UM 耐药和肝转移最重要的因素之一。我们前期工作表明 UM 存在CSCs，证据指向表观遗传蛋白 EZH2 调节和维持 CSCs 干性功能。本课题拟进一步证实 EZH2是调节和维持 UM CSCs 干性的重要蛋白。从 CSC的标志和功能实验系统观察高表达或抑制 EZH2 对CSCs 生物学行为的影响；对干细胞相关信号转导途径的影响；对 UM 细胞转移和侵袭能力的影响；检测化学抑制EZH2对 UM 肿瘤干细胞的杀伤效应。通过上述研究为 UM 找到治疗靶点和清除 UM 肿瘤干细胞的一种方法，为治疗 UM 提供转化医学依据。

脉络膜黑色素瘤 (uveal melanoma, UM) 是最常见的成人眼内肿瘤。约50%的 UM 病人发生肝转移。目前没有有效的治疗措施。约80% 的 UM 病人存在 GNAQ/GNA11 突变。BAP1 失活突变预示着 UM 病人有高转移风险。但 UM 发生肝转移的机制不清楚。表观遗传演变在循环肿瘤细胞 (circulating tumor cells, CTCs) 转移至远端器官的过程中发挥了重要的作用。我们提出假设，在 UM 细胞的转移过程中，表观遗传关键分子 EZH2 促使UM 细胞获得干性和增强的运动能力等恶性表型。本课题旨在研究确认 EZH2 作为转移性 UM 病人的潜在靶点。用 qRT-PCR, Western blotting 和免疫组化染色等方法检测了UM 病人样本和细胞系中 EZH2 的表达水平。继而通过 shRNA 干扰 EZH2 的表达或 EZH2 小分子抑制剂 GSK126 抑制 EZH2 的活性，以检测 UM 细胞的增殖、凋亡、肿瘤干细胞样特征、转移和侵袭能力。用 NOD/SCID 小鼠皮下移植瘤模型和 PDX 模型评价 GSK126 的体内抗肿瘤活性和肿瘤干细胞样细胞频率。用 NOG 小鼠肝转移模型评价 EZH2 促进 UM 细胞发生肝转移的能力和 GSK126 抑制 UM 细胞发生肝转移的活性。结果表明，与正常脉络膜组织相比，EZH2 在 UM 细胞和病人组织中高表达；EZH2 能促进 UM 肿瘤生长；EZH2 通过调控 miR-29c-DVL2-β-catenin 信号轴增加肿瘤干细胞样细胞频率；EZH2 通过调控 RhoGDIγ-Rac1 信号通路促进 UM 细胞迁移和侵袭。用 shRNA 或特异性小分子抑制剂 GSK126 能清除肿瘤干细胞样细胞，抑制 UM 细胞的运动能力，从而阻止 UM 发生肝转移。本课题的研究确认 EZH2 是治疗转移性 UM 病人的潜在靶点，为 GSK126 用于治疗转移性 UM 提供转化医学证据。