FAT10直接稳定HIF1α调控其表达促进肿瘤血管新生的机制研究

Our study had demonstrated that FAT10 was highly expressed in tumor and stabilized the substrate to promote tumor invasion and metastasis (Cancer Research, 2014 and 2016). The main cause of tumor invasion and metastasis was angiogenesis. HIF1α plays an important role in angiogenesis, and its E3 enzyme VHL promotes ubiquitination degradation of HIF1α to inhibit tumor angiogenesis. However, the relationship between FAT10 and HIF1α remains unclear. Our previous study found that, in VHL+/+ tumor cells and animal models, knockdown of FAT10 decreased HIF1α expression to inhibit tumor angiogenesis. The result of Co-IP had showed the interaction between FAT10 and HIF1α. Inhibition of proteasome degradation could block FAT10-mediated regulation of HIF1α expression. However, the above phenomenon was also found in VHL-/- tumor cells, suggested that FAT10 inhibited the degradation of HIF1α via E3 enzyme VHL-independent pathway. In summary, we speculated that FAT10 directly stabilizes HIF1α expression and represses its ubiquitination degradation to promote tumor angiogenesis. In order to verify the hypothesis, in this study, its specific mechanisms will be explored at the animal, cellular and molecular levels by using nude mouse tumorigenicity assay and in vitro ubiquitination assay. This study will provide a new theoretical basis for inhibiting tumor angiogenesis.

我们发现FAT10在肿瘤中高表达并稳定底物从而促进肿瘤侵袭转移（Cancer Research, 2014，2016）。肿瘤发生侵袭转移主要原因是血管新生，而HIF1α在调控血管新生中起重要作用，其E3酶VHL促进HIF1α经泛素降解从而抑制血管新生。但FAT10与HIF1α间关系尚不清楚。前期在VHL+/+肿瘤细胞和动物模型中发现：干扰FAT10导致HIF1α表达下降从而抑制肿瘤血管新生；免疫共沉淀结果发现FAT10与HIF1α结合；抑制蛋白酶体降解可阻断FAT10对HIF1α的调控。而在VHL-/-肿瘤细胞也发现上述现象，提示FAT10抑制HIF1α泛素化降解不依赖VHL。综上，我们推测FAT10通过直接稳定HIF1α的表达抑制其泛素化降解从而促进肿瘤血管新生。为验证假说，本课题拟采用裸鼠成瘤、体外泛素化等实验从动物、细胞和分子等水平探讨其具体机制，为抑制肿瘤血管新生提供新的理论依据。

肿瘤发生侵袭转移主要原因是血管新生，而HIF1α在调控血管新生中起重要作用，其E3酶VHL促进HIF1α经泛素降解从而抑制血管新生。研究发现类泛素蛋白FAT10在许多恶性肿瘤中高表达，FAT10通过稳定或降解底物从而促进肿瘤侵袭转移。在本研究中，我们在 A498（HA-VHL）和 A498（VHL-/-）细胞中分别转染shFAT10 质粒和Flag-FAT10 质粒后，检查 FAT10、HIF1a、VHL和VEGF 蛋白表达情况，结果发现：在 A498（HA-VHL）细胞中，降低 FAT10 的表达，VHL 表达增加，HIF1a 和 VEGF 蛋白表达下降，相反，过表达 FAT10，VHL 表达下降，HIF1a和VEGF蛋白表达增加。在A498（VHL-/-）细胞中，降低FAT10的表达，HIF1a 和 VEGF 蛋白表达下降，相反，过表达 FAT10，HOXB9，HIF1a 和VEGF蛋白表达增加。这些结果进一步证实 FAT10 调控 HIF1a 蛋白的表达不依赖VHL。我们研究还发现降低 FAT10后，PHD2蛋白表达增加，VHL蛋白表达也随之增加。这些结果提示FAT10也可能通过调控PHD2和VHL蛋白表达从而导致HIF-1a 蛋白表达增加。我们采用了免疫共沉淀等实验分析，结果表明FAT10肿瘤细胞中的HIF1α结合。GST-Pull down分析显示，FAT10与泛素竞争结合HIF1a，从而导致FAT10–HIF1α复合物的水平增加，Ub-HIF1α复合物的水平。本项目研究成果为抑制肿瘤血管新生提供新的理论依据。