RANKL promotes migration and invasion of hepatocellular carcinoma cells via NF-κB-mediated epithelial-mesenchymal transition

PLoS One. 2014 Sep 30;9(9):e108507. doi: 10.1371/journal.pone.0108507. eCollection 2014.

Abstract

Background: Metastasis accounts for the most deaths in patients with hepatocellular carcinoma (HCC). Receptor activator of nuclear factor kappa B ligand (RANKL) is associated with cancer metastasis, while its role in HCC remains largely unknown.

Methods: Immunohistochemistry was performed to determine the expression of RANK in HCC tissue (n = 398). Quantitative real-time polymerase chain reaction (qRT-PCR) and Western blot were used to examine the expression of RANK, E-cadherin, N-cadherin, vimentin, Snail, Slug, Twist and MMPs in HCC cells. Wound healing and Transwell assays were used to evaluate cell migration and invasion ability.

Results: We found that expression of RANK, the receptor of RANKL, was significantly higher in HCC tumor tissues than in peritumor liver tissues (p<0.001). Constitutive expression of RANK was detected in HCC cell lines, which can be up-regulated when HCC cells were stimulated with RANKL. Notably, in vitro experiments showed that activation of RANKL-RANK axis significantly promoted migration and invasion ability of HCC cells. In addition, RANKL stimulation increased the expression levels of N-cadherin, Snail, and Twist, while decreased the expression of E-cadherin, with concomitant activation of NF-κB signaling pathway. Moreover, administration of the NF-κB inhibitor attenuated RANKL-induced migration, invasion and epithelial-mesenchymal transition of HCC cells.

Conclusions: RANKL could potentiate migration and invasion ability of RANK-positive HCC cells through NF-κB pathway-mediated epithelial-mesenchymal transition, which means that RANKL-RANK axis could be a potential target for HCC therapy.

Publication types

  • Research Support, Non-U.S. Gov't

MeSH terms

  • Antigens, CD / genetics
  • Antigens, CD / metabolism
  • Antineoplastic Agents, Phytogenic / pharmacology
  • Cadherins / agonists
  • Cadherins / antagonists & inhibitors
  • Cadherins / genetics
  • Cadherins / metabolism
  • Carcinoma, Hepatocellular / genetics*
  • Carcinoma, Hepatocellular / metabolism
  • Carcinoma, Hepatocellular / pathology
  • Carcinoma, Hepatocellular / surgery
  • Cell Line, Tumor
  • Cell Movement / drug effects
  • Collagenases / genetics
  • Collagenases / metabolism
  • Diffusion Chambers, Culture
  • Epithelial-Mesenchymal Transition / drug effects
  • Gene Expression Regulation, Neoplastic*
  • Humans
  • Liver Neoplasms / genetics*
  • Liver Neoplasms / metabolism
  • Liver Neoplasms / pathology
  • Liver Neoplasms / surgery
  • NF-kappa B / agonists
  • NF-kappa B / antagonists & inhibitors
  • NF-kappa B / genetics*
  • NF-kappa B / metabolism
  • Neoplasm Invasiveness
  • Nuclear Proteins / agonists
  • Nuclear Proteins / genetics
  • Nuclear Proteins / metabolism
  • RANK Ligand / genetics*
  • RANK Ligand / metabolism
  • RANK Ligand / pharmacology
  • Receptor Activator of Nuclear Factor-kappa B / genetics*
  • Receptor Activator of Nuclear Factor-kappa B / metabolism
  • Sesquiterpenes / pharmacology
  • Sesquiterpenes, Guaiane
  • Signal Transduction
  • Snail Family Transcription Factors
  • Transcription Factors / agonists
  • Transcription Factors / genetics
  • Transcription Factors / metabolism
  • Twist-Related Protein 1 / agonists
  • Twist-Related Protein 1 / genetics
  • Twist-Related Protein 1 / metabolism
  • Vimentin / genetics
  • Vimentin / metabolism

Substances

  • Antigens, CD
  • Antineoplastic Agents, Phytogenic
  • CDH2 protein, human
  • Cadherins
  • NF-kappa B
  • Nuclear Proteins
  • RANK Ligand
  • Receptor Activator of Nuclear Factor-kappa B
  • SNAI1 protein, human
  • Sesquiterpenes
  • Sesquiterpenes, Guaiane
  • Snail Family Transcription Factors
  • TNFRSF11A protein, human
  • TNFSF11 protein, human
  • TWIST1 protein, human
  • Transcription Factors
  • Twist-Related Protein 1
  • Vimentin
  • helenalin
  • Collagenases

Grants and funding

This work was supported by the Major Program of NSFC (No. 81030038), National Key Sci-Tech Project (2012ZX10002011), National Natural Science Foundation of China (Nos. 81272730, 81272725, 81372648), FANEDD (No. 201183), Shanghai “Promising Youth Medical Worker” Project (No. 13Y055), and the Fok Ying-Tong Education Foundation (No. 132029). The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript.