候选抑癌基因Ovol2调控鼻咽癌干细胞干性及EMT的作用机制

Nasopharyngeal carcinoma (NPC) is a highly malignant tumor prevalent in South China. The most common cause of treatment failure of NPC is recurrence and metastasis. The existence of cancer stem cells (CSCs) or stem-like cancer cells (SLCCs) is regarded as the cause of recurrence and metastasis. By gene expression microarray analysis we found that transcriptional factor Ovol2, a candidate tumor suppressor gene, may play an important role in the regulation of CSC and epithelial-mesenchymal transition (EMT). Ovol2 was reported to regulate epidermic cell proliferation and differentiation. However whether and how Ovol2 participates in the tumorigenesis hasn't been studied yet. Considering more than 90% of NPC cases are undifferentiated carcinoma, we infer that Ovol2 takes part in NPC tumorigenesis through regulating CSC self-renewal and differentiation and EMT. Next we validated its lower expression in NPC tissue and cell lines, compared to normal nasopharyngeal (NP) tissue and transformed NP epithelial cell line. Furthermore overexpression of Ovol2 in NPC cells reduces the self-renewal capability. Taken together, it is indicated that Ovol2 is highly related to NPC development and participates in the regulation of CSC. Thus we plan to study whether and how Ovol2 regulates stemness of CSC and EMT in NPC. First, we'll test whether Ovol2 can regulate self-renewal of CSC and EMT by over-expression and knockdown of Ovol2 in NPC cells. Secondly, using mouse model we'll investigate whether Ovol2 can impair tumor formation and metastasis efficiency in vivo. Thirdly, the key factors and pathways downstream of Ovol2 will be studied. Also, the protein level of Ovol2 and related factors in NPC tissue will be tested by IHC and together with clinical information we'll elucidate the relationship between Ovol2 and tumor stage, recurrence, metastasis and prognosis by statistical analysis. These studies will reveal the molecular basis and clinical significance of Ovol2 in NPC development and help us to understand the regulatory mechanism of NPC stem cell and NPC recurrence and metastasis.

鼻咽癌是高发于中国华南地区的恶性肿瘤，复发和转移是限制预后和治疗失败的首要因素，而肿瘤干细胞被认为是恶性肿瘤复发和转移的根源。我们通过基因差异表达芯片筛选出和鼻咽癌干细胞、EMT相关的候选抑癌基因Ovol2。有研究表明该基因参与表皮细胞增殖和分化调节，但其和肿瘤关系未见报导。由于90%以上鼻咽癌是未分化型上皮细胞癌。因此我们推测Ovol2通过调节鼻咽癌干细胞以及EMT影响肿瘤发生发展。细胞和肿瘤组织预实验进一步表明Ovol2低表达和鼻咽癌进展相关；而其过表达则降低鼻咽癌细胞自我更新能力和β-Catenin表达。我们下一步拟检测该基因对肿瘤干细胞干性、分化及EMT的影响；并利用动物模型检测其对成瘤效率和肿瘤转移的影响；分析其参与干性调控和EMT的相关通路；同时在大量临床标本中验证其和肿瘤分期、复发、转移等相关性，阐明临床意义。该研究将揭示Ovol2参与鼻咽癌发生发展的分子机理和临床意义。

鼻咽癌是起源于鼻咽上皮的一种恶性肿瘤，高发于我国华南地区以及东南亚，发病具有明显的家族聚集性。由于90%以上属于未分化非角化型鼻咽癌，诱导鼻咽癌细胞分化是潜在的鼻咽癌治疗手段。在本项目资助下，我们探讨了转录因子Ovol2在鼻咽癌分化中的功能以及其相关的下游通路。首先，我们发现鼻咽癌肿瘤组织中Ovol2显著下调，并且在肿瘤组织和鼻咽癌细胞系中存在启动子区域的DNA甲基化。Ovol2过表达抑制鼻咽癌细胞在裸鼠体内的成瘤效率和生长速度；在体外抑制肿瘤细胞克隆形成；而Ovol2敲除的细胞克隆在裸鼠和体外均具有更强的克隆形成能力。进而，我们发现Ovol2的过表达和敲除分别伴随上皮细胞分化标志物deltaNP63以及deltaNP63靶基因的上调和下调。在Ovol2敲除细胞中恢复deltaNP63的表达，可以回复deltaNP63靶基因的表达，但是并不能完全恢复细胞分化，提示Ovol2促鼻咽癌分化的功能仅部分通过deltaNP63实现。最后，我们使用分化诱导剂全反式维甲酸（RA））诱导鼻咽癌细胞分化，可以观察到Ovol2表达增加；在Ovol2敲除细胞中，RA诱导分化效果下降；提示RA诱导分化部分通过增强Ovol2表达实现。并且在鼻咽癌细胞中使用DNA甲基化抑制剂5-Aza，能够促进Ovol2表达和分化。本课题首次揭示了Ovol2在鼻咽癌中诱导分化的重要作用，其下调能够促进鼻咽癌发生。我们的研究有助于理解鼻咽癌发生，特别是其未分化特性，提示了Ovol2可作为鼻咽癌标记物和潜在的治疗靶点。