FOXM1非转录依赖抑制人端粒酶逆转录酶泛素化降解促胃癌侵袭转移的作用及机制研究

As a nuclear transcription factor, FOXM1 plays an important role in the metastasis of gastric cancer. Recent study and our research found that FOXM1 also promotes the invasion and metastasis of gastric cancer independent of transcription function, but the mechanism is not clear. We demonstrated that the expression of FOXM1 positively correlates with hTERT in gastric cancer tissue, and either of them indicates poor prognosis. Recovery experiments showed that FOXM1 promotes metastasis of gastric cancer by up-regulating hTERT. Furthermore, protein interaction and co-localization between FOXM1 and hTERT were confirmed by Co-IP and immunofluorescence, respectively. Dual luciferase assays and protein stability experiments indicated that FOXM1, independent of transcriptional activity, inhibits ubiquitin degradation of hTERT. Mass spectrometry analysis indicated the potential binding of hTERT to E3 ubiquitin ligase. Interestingly, bioinformatics predicted that the binding site of hTERT protein to potential E3 ubiquitin ligase overlaps with that to FOXM1, so it is proposed that "FOXM1 competes E3 ubiquitin ligase for the binding to hTERT, thereby inhibiting the ubiquitination degradation of hTERT, which promotes invasion and metastasis of gastric cancer". This project intends to elucidate the molecular mechanism of FOXM1 mediated inhibition of hTERT ubiquitination by Co-IP, GST-pull down, FRET and other molecular techniques, and provides a new target for the development of anti-cancer drugs.

FOXM1作为核转录因子在肿瘤侵袭中发挥重要作用。文献及我们发现FOXM1还可依赖非转录活性促进胃癌侵袭，但机制不明。预实验发现胃癌组织中FOXM1与hTERT呈正相关，并与患者预后负相关；回复实验表明FOXM1通过上调hTERT促胃癌侵袭；Co-IP和共聚焦证实FOXM1与hTERT存在蛋白互作及共定位；双萤光素酶及蛋白稳定性实验发现FOXM1非转录依赖可抑制hTERT泛素化降解；质谱提示hTERT可与E3泛素连接酶潜在结合；生物信息学预测发现hTERT蛋白上E3泛素连接酶结合位点与FOXM1结合域重合。据此提出“FOXM1竞争某E3泛素连接酶与hTERT的结合，从而抑制hTERT泛素化降解，进而促进胃癌侵袭转移”的假说。本项目拟采用Co-IP,GST-pull down,FRET等技术，阐明FOXM1非转录活性抑制hTERT泛素化降解的分子基础，为靶向FOXM1抗癌药物研发提供新靶点。

FOXM1作为核转录因子在肿瘤侵袭中发挥重要作用。文献及我们的研究发现FOXM1还可依赖非转录活性促进胃癌侵袭，但机制不明。预实验发现胃癌组织中FOXM1与hTERT呈正相关，并与患者预后负相关；回复实验表明FOXM1通过上调hTERT促胃癌侵袭；Co-IP和共聚焦证实FOXM1与hTERT 存在蛋白互作及共定位；双萤光素酶及蛋白稳定性实验发现FOXM1非转录依赖可抑制hTERT泛素 化降解；质谱提示hTERT可与E3泛素连接酶潜在结合；生物信息学预测发现hTERT蛋白上E3泛素连接酶结合，位点与FOXM1结合域重合。依据已有文献的报道，我们首先筛查已知的hTERT泛素酶，发现FOXM1可以干扰其中MKRN1与hTERT的结合，而对MDM2的结合没有影响。据此提出“FOXM1竞争MKRN1与hTERT的结合， 从而抑制hTERT泛素化降解，进而促进胃癌侵袭转移”的结论。本项目采用Co-IP,GST-pull down,FRET等技术，阐明FOXM1非转录活性抑制hTERT泛素化降解的分子基础，为靶向FOXM1抗癌药物研发提供新靶点。