Axin-RhoBTB3-VHL复合体调控HIF1信号通路的分子机制及其生物学功能

The hypoxia-inducible factor (HIF1α) signal pathway plays very important roles in tumor metabolism, which promotes tumor survival and development by regulating its downstream target genes such as GLUT1, HK, LDH, and VEGF expresstion. These target genes are cloasely related to metabolism. We have employed several methods such as yeast two-hybrid screen, co-immunoprecipitation, subcellular co-localization to identify additional complexes centered on Axin, revealing a novel complex comprised of Axin, RhoBTB3, and VHL. We found that RhoBTB3 is significantly downregulated and HIF1α signal is upregulated in kidney cancer samples in the Oncomine. Knockdown of RhoBTB3 in Hela cells promoted xenograft growth in nude mice. Moreover, overexpressed RhoBTB3 in cells could decrease protein levels of HIF1α, which could be blocked by prior MG132 treatment. We hereby propose to explore the molecular mechanism and the effect on tumor metabolism of regulation HIF1α protein levels by the Axin-RhoBTB3-VHL complex under hypoxic conditions using immunoprecipitation, immunostaining, real-time quantitative PCR and etc. Emphasis will placed on how the complex is assembled and the mechanism of the dynamic regulation of the complex. Our study will make major contributions to the understanding of the regulation of the important factor HIF1α, and the mechanism of the tumor suppression by VHL.

低氧诱导因子HIF1α信号通路对肿瘤细胞的代谢与生长有着非常重要的影响。它通过正调节下游基因如GLUT1、HK、LDH、VEGF的表达，促进肿瘤的生存和发展。我们通过包括酵母双杂交系统、免疫共沉淀、细胞共定位多种方法，鉴定了Axin-RhoBTB3-VHL这个新的复合体。我们发现，RhoBTB3在肾癌样本文库中出现显著下调，而HIF1α的下游基因出现相应上调；用siRNA敲低Hela细胞的RhoBTB3, 并注射裸鼠，可以促进裸鼠移植瘤的生长；过表达RhoBTB3对于HIF1α的蛋白水平具有下调作用，而MG132处理可以抑制这种下调作用。我们拟通过免疫共沉淀、免疫荧光、实时定量PCR等实验探索低氧刺激下Axin-RhoBTB3-VHL复合体调节HIF1α蛋白水平的分子机制及其对肿瘤细胞生长和代谢的影响。

低氧诱导因子(HIFs)信号通路在肿瘤代谢中起着非常重要的作用，HIFs的异常积累或激活和许多类型的癌症密切相关。然而，HIF1α蛋白如何被调控（包括羟基化和泛素化）的分子机制目前还不清楚。我们在研究中发现，Rho GTPase蛋白RHOBTB3可直接与羟基化酶PHD2相互作用，从而促进HIF1α的羟基化。此外，RHOBTB3也可直接与E3泛素连接酶的重要组成分子VHL蛋白相互作用，促进了HIF1α的泛素化。值得注意的是，我们还发现RHOBTB3可与已知的HIF1α调控因子LIMD1形成二聚体，并组成一个包含RHOBTB3/LIMD1-PHD2-VHL-HIF1α的复合体，从而达到最大限度地降解HIF1α。细胞在缺氧条件下降低了以RHOBTB3为核心的复合体的形成，继而导致HIF1α的积累。这些发现帮助我们更好地认知HIF1α的调节机制，也揭示了肿瘤细胞面对低氧环境发生应激改变而获得更好的生长的重要分子机制，为治疗肿瘤提供了新的思路和药物设计靶点。