胶质瘤干细胞中MTDH蛋白维持FoxM1高表达并激活FoxM1转录的分子机制研究

Previously we had identified FoxM1 plays a pivotal role in maintaining Glioma Stem Cell (GSC) self-renewal. Knocking down FoxM1 could reduce the tumorigenecity in GSC significantly. However, transient over-expression of FoxM1 in G2/M phase is required for the mitosis process in normal glia cells. Unselective inhibition of FoxM1 could induce permanent and irreversible damages to normal glia cell while killing GSC. Research showed that over-expression of FoxM1 in GSC was caused by the N-terminal induced ubiquitination devitalization. By using LC/LC Mass Spectrum, we identified a novel FoxM1 N-terminal binding protein MTDH, which is highly expressed in GSC but no detectable expression in normal glia cells, may be involved in the deactivation mechanism of FoxM1 ubiquitination. Deprivation of MTDH in GSC could increase FoxM1 ubiquitination induced degradation and thus inhibits GSC self-renewal and tumorigenecity. In this application, we plan to study the biochemical mechanisms of MTDH/FoxM1 interaction; molecular mechanisms of MTDH maintain FoxM1 over-expression through deactivating its ubiqutination degradation and molecular mechanisms of MTDH activates FoxM1 transcription in GSC. Though these studies we hope to provide theoretical evidences to make MTDH an ideal molecular target in GSC treatment.

前期研究证实，FoxM1对维持胶质瘤干细胞（GSC）的自我更新能力具有重要作用；敲除FoxM1可以有效抑制GSC的致瘤性。然而，FoxM1在G2/M期一过性表达增加是正常胶质细胞有丝分裂必需，非选择性的抑制FoxM1在杀灭GSC的同时也对正常胶质细胞造成不可逆损伤。研究表明，FoxM1在GSC内的高表达是其蛋白N末端介导的泛素化降解机制失活导致。我们利用液相质谱技术发现一种在GSC高表达的而正常胶质细胞不表达的原癌基因MTDH结合在FoxM1蛋白N末端，可能与FoxM1泛素化降解机制失活有关。敲除MTDH后可以增加FoxM1泛素化降解，抑制GSC的自我更新能力和致瘤性。本申请拟阐明MTDH在GSC内结合FoxM1并抑制其泛素化降解、维持其蛋白稳定性以及转录活性，从而导致GSC自我更新和恶性胶质瘤发生发展的详细分子机制，并在动物模型中验证，为将MTDH作为更理想的GSC治疗靶点提供理论依据。

本项目前期研究证实FOXM1在胶质瘤中过表达并具有重要作用；敲除FOXM1可以有效地在体内外抑制胶质瘤生长，但FOXM1在G2/M期一过性表达增加是正常胶质细胞有丝分裂必需，非选择性的抑制FOXM1在杀灭胶质瘤的同时也对正常胶质细胞造成不可逆损伤。在本研究中首次证实了在恶性胶质瘤中FOXM1受到MTDH表达的直接调控,并详细阐明了MTDH通过与FOXM1的N端结合，竞争抑制了CDH1结合在FOXM1上。由于FOXM1的蛋白水平被认为部分受控于E3泛素化连接酶APC/C。APC/C在有丝分裂结束的时候介导了FOXM1的降解，而在这个过程依赖于CDH1对于FOXM1 N端的D-BOX和KEN-BOX的识别。由此MTDH-FOXM1的相互作用对于CDH1的竞争抑制防止了FOXM1在随后的蛋白酶体降解。删除了FOXM1的MTDH结合位点DMBS则消除了FOXM1由于MTDH过表达介导的稳定性。MTDH同时也结合到FOXM1的靶基因上促进并增强FOXM1的转录活性。敲低MTDH使FOXM1变得不稳定并降低其转录活性,导致细胞周期进程、血管生成及肿瘤细胞在体内体外的侵袭能力被抑制。本研究在分子水平详细阐释了MTDH和FOXM1在人类恶性胶质瘤中高表达的原因以及相关性,并进一步在动物模型中验证了这一结论。研究发现并阐明的MTDH在胶质瘤进程中正向调控FOXM1稳定性和功能的原理和机制为临床上治疗恶性胶质瘤提供了潜在的治疗靶点和新的治疗策略。