Login to Access Video or Poster Abstract: MP87-16
Sources of Funding: DoD postdoc training award (W81XWH-14-1-0249)_x000D_ Urology Care Foundation Research Scholar Award

Introduction

Despite the role of interferon-γ (IFNγ) in tumor immune surveillance; studies have implicated the dark side of IFNγ based on its pro-tumorigenic activity. IFNγ can induce transcriptional activation of IFN-stimulated genes (ISGs) via JAK-STAT signaling pathway. The most highly induced ISGs are interferon-induced tetratricopeptide repeat (IFIT) family members. By studying a differential regulation on a unique tumor suppressor miR-363 from its polycistronic miR-106a-363 cluster, we unveiled a new microRNA (miRNA) turnover machinery composed of IFIT5, which is first described as a viral RNA binding protein. Up to date, the impact of IFIT5 on cancer metastasis is unclear.

Methods

Luciferase reporter gene assay was for examining the IFNγ-induced IFIT5 gene activation. Transwell migration assay was for demonstrating the function of IFIT5 with cancer metastasis. Site-directed mutagenesis, in vitro transcription, RNA pull down and in vitro RNA degradation assay were for determining the specificity of miRNA species regulated by IFIT5-mediated turnover machinery.

Results

IFIT5 gene promoter activity and protein level were significantly elevated by IFNγ. IFIT5 complex represents unique post-transcriptional machinery for turnover of a specific population of tumor suppressor miRNAs. We examined several IFIT5-regulated miRNA candidates, and found IFNγ can suppress miR-101, miR-335, miR-203, and miR-128, and phenocopied the miRNA expression profile of IFIT5-overexpressing cells. Seed regions of miR-101 and miR-128 have sequence-matched target sites at ZEB1 mRNA 3&[prime]UTR, and indeed can suppress ZEB1. Meanwhile, IFIT5 is elevated in higher grade prostatic tumors, and positively correlated with ZEB1, ZEB2 and Slug in prostate cancer. Knockdown of IFIT5 lead to suppression of ZEB1 and Slug, along with decreased migration mobility in cancer cells. On the contrary, IFNγ treatment enhanced cell migration, and this effect is diminished by the loss of IFIT5. We also modified the 5&[prime]end structure of precursor miR-101 and miR-128, and examined its regulation by IFIT5-mediated miRNA turnover machinery. Both pre-miR-101 and pre-miR-128 with mutated blunt end are resistant to degradation in an IFIT5-expressing PC3 cell and show greater impact on suppressing cell migration, compared to the mutant precursor with single stranded overhang.

Conclusions

We demonstrated that IFNγ potentiate prostate cancer metastasis via IFIT5-mediated miRNA turnover machinery, which regulates specific tumor suppressor miRNAs that target critical EMT factors including ZEB1 and Slug._x000D_ _x000D_

Funding

DoD postdoc training award (W81XWH-14-1-0249)_x000D_ Urology Care Foundation Research Scholar Award