NF-κB-Induced Upregulation of miR-548as-3p Increases Invasion of NSCLC by Targeting PTEN.

BACKGROUND: Non-Small Cell Lung Cancer (NSCLC) is an aggressive cancer type due to high metastatic capacity. Nuclear Factor Kappa B (NF-κB) is a consistently active transcription factor in malignant lung cancer cells and has crucial significance in NSCLC progression. It is also implicated in the transcriptional regulation of many genes including microRNAs (miRNAs) that function as tumor suppressor or oncogene. It has been increasingly reported that several miRNAs defined as gene members are induced by NF-κB. The present study aimed to find novel miRNAs that are regulated by NF-κB. METHODS: Chromatin Immunoprecipitation Sequencing (ChIP-Seq) experiment and bioinformatic analysis were used to determine NF-κB-dependent miRNAs. Western blot analysis, quantitative real-time polymerase chain reaction (qRT-PCR), luciferase reporter gene assays were carried out to investigate the target genes of miRNAs. To determine biologic activity, transwell invasion and MTT assay were carried out on H1299 NSCLC cell line. miRNA expression level was evaluated in metastatic and non-metastatic tissue samples of NSCLC patients. RESULTS: ChIP-Seq and qRT-PCR experiments showed that miR-548as-3p is transcriptionally regulated by NF- κB in response to Tumor Necrosis Factor-α (TNF-α) treatment. Then, we found that tumor suppressor Phosphatase and Tension homolog (PTEN) is a direct target of miR-548as-3p. Furthermore, miR-548as-3p mediates phosphatidylinositol-3-OH kinase (PI3K)/Akt pathway and NF-κB-implicated genes including Matrix Metalloproteinases 9 (MMP9), Slug and Zeb1. We further showed that miR-548as-3p increased invasiveness of NSCLC cells and was upregulated in metastatic tumor tissues compared to non-metastatic ones. CONCLUSION: All these findings provide a miRNAs-mediated novel mechanism for NF-κB signaling and that miR-548as-3p could be a biomarker for NSCLC metastasis.

Protein kinase A-mediated phosphorylation regulates STAT3 activation and oncogenic EZH2 activity.

Polycomb repressive complex 2 (PRC2) member enhancer of zeste homolog 2 (EZH2) trimethylates histone H3 lysine 27 (H3K27me3), alters chromatin structure and contributes to epigenetic regulation of gene expression in normal and disease processes. Phosphorylation of EZH2 augmented EZH2 oncogenic activity in cancer but observations have been limited to threonine 350 (T350) and serine 21 (S21) residues by cyclin-dependent kinase 1 and protein kinase B, respectively. In addition, phosphorylation of the evolutionarily conserved T372 motif of EZH2 by p38 resulted in EZH2 interaction with Ying Yang 1 and promoted muscle stem cell differentiation. In the present study, we used epithelial ovarian cancer (OC) cells as a model to demonstrate that phosphorylation of EZH2 at T372 by protein kinase A (PKA) induced a dominant-negative EZH2 phenotype, inhibited OC cell proliferation and migration in vitro and decreased ovarian xenograft tumor growth in vivo. Phosphorylation of T372 by PKA enhanced the interaction between EZH2 and signal transducer and activator of transcription 3 (STAT3), and STAT3 binding to pT372-EZH2 reduced cellular levels of pSTAT3 and downregulated interleukin 6 receptor expression in OC. Furthermore, PKA-mediated pT372-EZH2 decreased ATP levels and altered mitochondrial gene expression, resulting in mitochondrial dysfunction and reduced OC cell growth. These findings demonstrate that PKA-mediated T372 phosphorylation reduces oncogenic EZH2 activity and reveal a novel role for pT372 in regulating EZH2 in OC and possibly other cancers.

Cell type-specific expression of the IkappaB kinases determines the significance of phosphatidylinositol 3-kinase/Akt signaling to NF-kappa B activation.

Phosphatidylinositol (PI) 3-kinase/Akt signaling activates NF-kappa B through pleiotropic, cell type-specific mechanisms. This study investigated the significance of PI 3-kinase/Akt signaling to tumor necrosis factor (TNF)-induced NF-kappa B activation in transformed, immortalized, and primary cells. Pharmacological inhibition of PI 3-kinase blocked TNF-induced NF-kappa B DNA binding in the 293 line of embryonic kidney cells, partially affected binding in MCF-7 breast cancer cells, HeLa and ME-180 cervical carcinoma cells, and NIH 3T3 cells but was without significant effect in H1299 and human umbilical vein endothelial cells, cell types in which TNF activated Akt. NF-kappa B is retained in the cytoplasm by inhibitory proteins, I kappa Bs, which are phosphorylated and targeted for degradation by I kappa B kinases (IKK alpha and IKK beta). Expression and the ratios of IKK alpha and IKK beta, which homo- and heterodimerize, varied among cell types. Cells with a high proportion of IKK alpha (the IKK kinase activated by Akt) to IKK beta were most sensitive to PI 3-kinase inhibitors. Consequently, transient expression of IKK beta diminished the capacity of the inhibitors to block NF-kappa B DNA binding in 293 cells. Also, inhibitors of PI 3-kinase blocked NF-kappa B DNA binding in Ikk beta-/- but not Ikk alpha-/- or wild-type cells in which the ratio of IKK alpha to IKK beta is low. Thus, noncoordinate expression of I kappa B kinases plays a role in determining the cell type-specific role of Akt in NF-kappa B activation.