FOXM1非转录样效应抑制线粒体自噬促进胃癌侵袭转移的作用及分子机制研究

Invasion and metastasis are the leading cause of death in gastric cancer. FOXM1 plays an important role in it. The current mechanism is focused on FOXM1 serves as a transcription factor to directly regulate the target gene. However, previous studies show that FOXM1Mut, which lacks transcriptional activity, still promotes invasion and metastasis, and the mechanism is not clear. Mitophagy is closely related to tumor invasion and metastasis. In the present study, we found that FOXM1 inhibited mitophagy in gastric cancer cells, which is independent upon the transcriptional activity, and increasing this mitophagy significantly impaired the FOXM1/FOXM1Mut-induced metastasis. Subsequently, we discovered that, FOXM1/FOXM1Mut inhibited mitophagy mainly through suppressing BNIP3 protein stability, whereas increasing BNIP3 restored the FOXM1/FOXM1Mut-inhibited mitophagy. Moreover, we demonstrated that FOXM1/FOXM1Mut increased the ubiquitination of BNIP3.Furthermore, we verified that FOXM1 interacted with BNIP3, and LC-MS-MS indicated the existance of E3 ligase in FOXM1/BNIP3 complex. Taken together, these results suggests that, FOXM1 may mobilize an E3 ligase to bind with BNIP3, suppressing the expression of BNIP3 and mitophagy accordingly. Ultimately, FOXM1 promotes the invasion of gastric cancer cells independent on its transcriptional activity. We would like to further confirm the E3 liagase which mediated BNIP3 ubiquitination and resolve the ubiquitination sites in it using co-immunoprecipitation(Co-IP), GST-pull down, and fluorescence resonance energy transfer(FRET). Our study may enrich the mechanism of FOXM1-induced metastasis in gastric cancer, providing a new target for FOXM1 drug development.

侵袭转移是胃癌死亡的主要原因,叉头盒蛋白M1(FOXM1)在其中发挥重要作用。目前主要研究是其作为转录因子直接调控转移相关基因表达,从而促侵袭转移。文献及课题组预实验表明无转录活性的FOXM1仍可促侵袭转移,但机制不清。预实验发现FOXM1可非依赖转录促BNIP3降解,抑制线粒体自噬促胃癌侵袭转移。生物信息学预测发现BNIP3存在潜在泛素化位点,预实验证实FOXM1显著促BNIP3泛素化,且存在蛋白互作及共定位,质谱结果表明FOXM1/BNIP3复合物中存在潜在E3泛素连接酶,提示FOXM1可能通过募集某E3,使之与BNIP3结合促其泛素化,通过非转录效应抑制线粒体自噬,促胃癌细胞侵袭转移。本项目拟采用Co-IP,GST-pull down,FRET确定介导BNIP3泛素化的E3,解析其导致BNIP3泛素化的具体位点,丰富FOXM1促胃癌侵袭转移的机制,为靶向FOXM1药物研发提供新靶点。

叉头盒蛋白M1(FOXM1)在肿瘤恶性进展中发挥重要作用，于 2010 年被分子和细胞生物学及生物技术方案和研究国际协会(ISMCBBPR)评为年度分子，提示其在癌症治疗方面具有巨大潜力。目前主要研究是FOXM1作为转录因子直接调控增殖及转移相关基因表达,从而促肿瘤进展。文献及课题组发现FOXM1可通过调节E3样泛素连接酶FBXW7，促进BNIP3泛素化降解，抑制线粒体自噬从而增强胃癌细胞侵袭。此外，在进行上述工作过程中，课题组还发现了可调控FOXM1的新分子PIN1，其高表达是导致胃癌中FOXM1异常增高的重要原因。而进一步解析发现miR628-5p是PIN1的重要调控分子，从而初步确立了miR628-5p-PIN1-FOXM1这一信号通路。此外，我们还发现了FOXM1的新修饰方式ISGylation，其可显著抑制FOXM1核转位，从而抑制胃癌细胞增殖及侵袭。上述发现不仅丰富了FOXM1促胃癌侵袭转移的机制,还可能为靶向FOXM1药物研发提供新靶点