FoxM1与Wnt通路协同调控GPR4促卵巢癌增殖转移及血管生成的分子机制研究

The treatment effect and the prognosis of ovarian cancer are very poor. In our previous study, we have firstly found that that GRP4 gene plays important roles in promoting the growth, metastasis, and the angiogenesis of ovarian cancer, as well as inhibiting its apoptosis. But the mechanism of GPR4 effect on the development of ovarian cancer is still unknown. Data of cDNA microarray and Wnt array we have previously obtained showed that,the expression of most members of Wnt signaling pathway have changed after the expression of GRP4 gene was knocked out, which indicating the Wnt/TCF signaling pathway probably mediates the effect of GPR4 on promoting the development of ovarian cancer. In our previous study, we have found the expression of β-catenin and TCF3 was high in ovarian cancer cells in which GPR4 overexpressed. When the expression of TCF-3 was knocked out, the promoting effect of GPR4 on ovarian cancer cells was significantly inhibited. FoxM1, a transcription factor, can regulate the function of β-catenin. How does the Wnt/TCF signaling pathway specifically mediate the function of GRP4? Hypothesis 1 we speculated is that, in ovarian cancer cells, FoxM1 combines with β-catenin and helps β-catenin to be transfered into cellular nuclear. And there they form transcription complex with TCF3, which binds to the promoter of GPR4 to increase its transcriptional activity; Furthermore, FoxM1 and ICAT can combine with β-catenin competitively. Besides, Wnt3a is highly expressed in ovarian cancer; Hypothesis 2 we speculated is that, Wnt3a regulated the GPR4 function through regulating the competitive inhibition of ICAT on the binding of FoxM1 with β-catenin; High level of intracellular FoxM1 is the prerequisite for driving the malignant behavior of tumor cells. But the level of FoxM1 changes dynamically during the cell cycle, and its level reach the lowest in the terminal stage. Based on our previous observation, the hypothesis 3 we speculated is that, Wnt3a regulates the expression of FoxM1 is through increasing the stability of FoxM1 during the cell cycles while without affecting the expression of ICAT. In this study we are going to prove these hypotheses and make clear the mechanism of GPR4 effect on the development of ovarian cancer.

卵巢癌疗效差。我们前期发现GPR4对卵巢癌生长迁移、血管生成及凋亡很重要。但机制不清楚。我们发现敲除GPR4后Wnt成员表达都改变，GPR4高表达细胞内β-catenin与TCF3也高表达，敲除TCF3后GPR4功能显著受抑制，说明Wnt可能介导GPR4的作用。Wnt具体怎样介导GPR4作用的？FoxM1可调节β-catenin功能。我们推测机制一：FoxM1与β-catenin结合后将其运至核内，并与TCF3结合，作用于GPR4启动子促其转录；ICAT和FoxM1可与β-catenin竞争性结合。Wnt3a在卵巢癌中高表达。我们推测机制二：Wnt3a通过调节ICAT的竞争性结合抑制来调节GPR4功能；FoxM1在细胞周期中呈动态变化。我们推测机制三：Wnt3a调节FoxM1表达是通过提高其在细胞周期中稳定性实现的，而对ICAT表达却无影响；拟验证这些假设以明确GPR4对卵巢癌作用的机制。

一. 在卵巢癌细胞中沉默FoxM1后发现：细胞增殖下降、凋亡增加、侵袭迁移下降、克隆形成减弱； G1期阻滞；干性成球减弱。FoxM1是个促癌基因。.在A2780中过表达GPR4后，Wnt信号通路被GPR4激活。.二. A2780中Wnt/β-catenin通路显著激活。敲低FoxM1后Wnt/β-catenin通路受到抑制。FoxM1与β-catenin均定位于细胞核内。发现FoxM1与β-catenin有空间重叠区域。FoxM1可能参与转运β-catenin入核的功能。.免疫共沉淀发现FoxM1可与β-catenin直接结合。说明FoxM1可通过与β-catenin直接结合来调节β-catenin的入核和功能。.分别构建7个β-catenin突变体，检测β-catenin与FOXM1结合位点，发现FoxM1与β-catenin的M4区域有结合..三. 抑制ICAT后，FoxM1与β-catenin结合明显增加。Axin-2和c-MYC的表达量均增高，说明β-catenin/TCF3转录活性明显增加。抑制ICAT可增加FOXM1与β-catenin的结合，并可增加β-catenin/TCF-3的转录活性。.四. 抑制FoxM1后，ICAT与β-catenin结合明显增加，下游Axin-2的表达量亦下降，说明FoxM1抑制ICAT与β-catenin的结合，并促进β-catenin/TCF-3的转录活性。.五. Wnt3a 可显著上调 FoxM1 的表达，并呈现剂量和时间依赖性。.Wnt3a对FOXM1的调节是通过促进FOXM1的稳定性实现的。.Wnt3a调节了FOXM1与ICAT之间的竞争机制。.六. FoxM1对GPR4的转录核心启动子元件位点:.在构建的4个GPR4启动子位点中， pGL6-GPR4-P1是FoxM1作用的启动子位点。.七. 在小鼠卵巢癌ID8中敲除FoxM1后肿瘤生长减慢，而敲除ICAT后肿瘤生长加快，肝脏发生转移，说明FoxM1促进卵巢癌生长、而ICAT抑制卵巢癌生长。.敲除FoxM1后，WNT3A表达上调、GPR4表达下降、β-catenin表达下降、TCF3表达下降。敲除ICAT这组，GPR4表达上调、β-catenin表达上调、TCF3表达上调；β-catenin主要在细胞浆；.八. 人卵巢癌组织FoxM1高表达与生存期呈负相关.