Abstract 1491: Downregulation of KLF5 contributes to TGF-β- and EGF-induced EMT by reducing miR-200s.

Abstract Krüpple-like factor 5 (KLF5) is a basic transcriptional factor that has been well documented for its regulatory role in the proliferation and survival of epithelial cells. Recently, bioinformatic analysis suggests that KLF5 also regulates epithelial-to-mesenchymal transition (EMT), yet experimental evidence is still lacking regarding whether and how KLF5 regulates EMT. As a form of epithelial cell plasticity, EMT can be induced by many cytokines including TGF-β and EGF. Here we report a role of KLF5 in EMT. We found that KLF5 was significantly down-regulated during TGF-β- and EGF-induced EMT in epithelial cells including HaCaT cells, MCF-10A cells, and mouse primary keratinocytes; and that silencing KLF5 induced EMT as well, as indicated by altered cell morphology, enhanced migration, upregulation of CDH2, FN1 and ZEB1, dislocation of CDH1 from cell-cell junctions, and induction of actin fibers. To determine whether miRNAs mediate the role of KLF5 in EMT, a real-time qPCR assay was employed to search for miRNAs regulated by KLF5 during EMT. Five members of the miR-200 family, all of which have been implicated in EMT, were found to be down-regulated upon the downregulation of KLF5. KLF5 directly regulated the expression of miR-200s by binding to the GC boxes of their promoters, as demonstrated by functional and biochemical analyses including luciferase reporter assay, ChIP-PCR assay, and oligo pull-down assay. Functionally, overexpression of miR-200s inhibited EMT induced by KLF5 silencing or TGF-β and EGF treatment in HaCaT cells, and forced expression of KLF5 attenuated TGF-β- and EGF-induced EMT by rescuing miR-200s expression. To evaluate whether the KLF5-miR-200s axis modulates EMT in vivo, we determined the expression of Klf5, miR-200s and EMT markers in knockout mice and human breast cancer specimens. In mouse prostate neoplasms induced by Pten deletion, knockout of Klf5 repressed the expression of miR-200s and induced the downregulation of CDH1 and upregualtion of vimentin, which are indicative of EMT. Expression of KLF5 also correlated with miR-200s at the RNA level in human breast cancer specimens. These findings suggest that KLF5 directly regulates the transcription of miR-200s in epithelial cells, and that downregulation of the KLF5-miR-200 axis plays a causal role in TGF-β- and EGF-induced EMT, providing new insights into the regulation of EMT. Citation Format: Baotong Zhang, Jin-tang Dong. Downregulation of KLF5 contributes to TGF-β- and EGF-induced EMT by reducing miR-200s. [abstract]. In: Proceedings of the 104th Annual Meeting of the American Association for Cancer Research; 2013 Apr 6-10; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2013;73(8 Suppl):Abstract nr 1491. doi:10.1158/1538-7445.AM2013-1491