RNF111/Arkadia is regulated by DNA methylation and affects TGF-β/Smad signaling associated invasion in NSCLC cells

Lung Cancer. 2015 Oct;90(1):32-40. doi: 10.1016/j.lungcan.2015.07.010. Epub 2015 Jul 26.

Abstract

Objectives: RNF111/Arkadia is a critical regulator of TGF-β signaling, being required for SMAD3-mediated responses such as TGF-β-induced repression of E-cadherin. Previous studies show that mutations in RNF111 in human cancers are rare and RNF111 promotes lung tumor metastasis. However, the epigenetic mechanisms underlying the role of RNF111 in non-small cell lung cancer (NSCLC) metastasis remain unknown. Here, we mainly focused on low- (95C) and high-metastatic (95D) NSCLC cell lines, which share a similar genetic background, and investigated the methylation-based regulation of RNF111 expression.

Materials and methods: Clonal bisulfite sequencing, real-time qRT-PCR, western blot analysis, luciferase reporter assays, RNA interference, chromatin immunoprecipitation (ChIP) assay and transwell migration and invasion assays were performed on human NSCLC cell lines 95C and 95D.

Results: RNF111 was high-expressed in 95D cells, which showed low-level methylation at -459CpG site in RNF111 promoter. The opposite results were obtained in 95C cells. Cell-based and biochemical assays revealed that -459CpG methylation can inhibit RNF111 transcriptional expression by interfering with the recruitment of Sp1 to RNF111 promoter. On TGF-β stimulation, siRNA-mediated RNF111 knockdown inhibited TGF-β/Smad signaling activity and Snail (an inducer of metastasis) expression, and enhanced E-cadherin (an epithelial-to-mesenchymal transition marker) expression in 95C and 95D cells. Furthermore, demethylation-induced upregulation of RNF111 enhanced phosphorylation of SMAD3 and Snail expression, and repressed E-cadherin expression in 95C cells expressing low RNF111.

Conclusions: Our results suggest that -459CpG methylation in Sp1-binding site of RNF111 promoter transcriptionally decreases RNF111 expression, which inhibits TGF-β/Smad signaling associated invasion in NSCLC cells.

Keywords: -459CpG methylation; Invasion; NSCLC; RNF111; TGF-β/Smad signaling.

MeSH terms

  • Carcinoma, Non-Small-Cell Lung / genetics
  • Carcinoma, Non-Small-Cell Lung / metabolism*
  • Carcinoma, Non-Small-Cell Lung / pathology
  • Cell Line, Tumor
  • DNA Methylation*
  • Epithelial-Mesenchymal Transition
  • Humans
  • Lung Neoplasms / genetics
  • Lung Neoplasms / metabolism*
  • Lung Neoplasms / pathology
  • Mutation
  • Neoplasm Invasiveness
  • Nuclear Proteins / genetics
  • Nuclear Proteins / metabolism*
  • Phosphorylation
  • Promoter Regions, Genetic
  • RNA Interference
  • RNA, Messenger / genetics
  • RNA, Messenger / metabolism
  • Smad3 Protein / genetics
  • Smad3 Protein / metabolism*
  • Transforming Growth Factor beta / genetics
  • Transforming Growth Factor beta / metabolism*
  • Ubiquitin-Protein Ligases / genetics
  • Ubiquitin-Protein Ligases / metabolism*
  • Up-Regulation

Substances

  • Nuclear Proteins
  • RNA, Messenger
  • SMAD3 protein, human
  • Smad3 Protein
  • Transforming Growth Factor beta
  • RNF111 protein, human
  • Ubiquitin-Protein Ligases