Previously, our laboratory demonstrated that a deregulated E2F5/p38/SMAD3 axis was associated with uncontrolled cellular proliferation in prostate cancer (PCa). Here we investigate the role of E2F5 in PCa in further details. RNAi-mediated E2F5 knockdown and pathway-focused gene expression profiling in PC3 cells identified TFPI2 as a downstream target of E2F5. Manipulation of E2F5 expression was also found to alter MMP-2 and MMP-9 levels as detected by Proteome-Profiler Array, western-blot and qRT-PCR. Site-directed mutagenesis, dual-luciferase assays and chromatin-immunoprecipitation with anti-E2F5-IgG coupled with qPCR confirmed recruitment of E2F5 on TFPI2, MMP-2 and MMP-9 promoters. RNAi-mediated knockdown of E2F5 expression in PC3 caused a significant alteration of cell migration while that of TFFI2 resulted in a modest change. Abrogation of E2F5 and TFPI2 expression was associated with significant changes in the gelatinolytic activity of active forms of MMP-2 and MMP-9. Moreover, E2F5, MMP-2 and MMP-9 levels were elevated in biopsies of PCa patients relative to that of benign hyperplasia, while TFPI2 expression was reduced. MMP-9 was co-immunoprecipitated with anti-TFPI2-IgG in PCa tissue samples suggesting a direct interaction between the proteins. Finally, Artemisinin treatment in PC3 cells repressed E2F5 along with MMP-2/MMP-9 while triggering TFPI2 expression which alleviated PC3 aggressiveness possibly through inhibition of MMP activities. Together, our study reinstates an oncogenic role of E2F5 which operates as a dual-function transcription factor for its targets TFPI2, MMP-2 and MMP-9 and promotes cellular invasiveness. This study also indicates a therapeutic potential of artemisinin, a natural compound which acts by correcting dysfunctional E2F5/TFPI2/MMP axis in PCa.

中文翻译：

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E2F5通过调节TFPI2，MMP-2和MMP-9促进前列腺癌细胞的迁移和侵袭。

以前，我们的实验室证明，E2F5 / p38 / SMAD3轴失控与前列腺癌（PCa）中不受控制的细胞增殖有关。在这里，我们进一步研究E2F5在PCa中的作用。RNAi介导的E2F5敲低和PC3细胞中以途径为中心的基因表达谱确定了TFPI2为E2F5的下游靶标。通过蛋白质组分析仪阵列，western印迹和qRT-PCR检测，还发现操纵E2F5表达可改变MMP-2和MMP-9水平。定点诱变，双重荧光素酶测定和抗E2F5-IgG的染色质免疫沉淀结合qPCR证实E2F5在TFPI2，MMP-2和MMP-9启动子上募集。RNAi介导的PC3中E2F5表达的敲低引起细胞迁移的显着变化，而TFFI2引起的细胞变化发生适度的变化。E2F5和TFPI2表达的废除与活性形式的MMP-2和MMP-9的明胶分解活性的显着变化有关。此外，相对于良性增生，PCa患者活检中E2F5，MMP-2和MMP-9水平升高，而TFPI2表达降低。在PCa组织样品中，MMP-9与抗TFPI2-IgG共免疫沉淀，表明这些蛋白之间存在直接相互作用。最后，在PC3细胞中用青蒿素处理可同时抑制E2F5和MMP-2 / MMP-9，同时触发TFPI2表达，从而可能通过抑制MMP活性来减轻PC3的侵袭性。总之，我们的研究恢复了E2F5的致癌作用，E2F5作为其靶标TFPI2，MMP-2和MMP-9的双重功能转录因子，并促进细胞侵袭性。