title: Activation of NF-κB by the RANKL/RANK system up-regulates snail and twist expressions and induces epithelial-to-mesenchymal transition in mammary tumor cell lines.
abstract: BACKGROUND
  Increased motility and invasiveness of cancer cells are reminiscent of the epithelial-mesenchymal transition (EMT), which occurs during cancer progression and metastasis. Recent studies have indicated the expression of receptor activator of nuclear factor-κB (RANK) in various solid tumors, including breast cancer. Although activation of the RANK ligand (RANKL)/RANK system promotes cell migration, metastasis, and anchorage-independent growth of tumor-initiating cells, it remains to be investigated if RANKL induces EMT in breast cancer cells. In this study, we investigated whether RANKL induces EMT in normal breast mammary epithelial cells and breast cancer cells, and the mechanism underlying such induction.
  
  
  METHODS
  Expression levels of vimentin, N-cadherin, E-cadherin, Snail, Slug, and Twist were examined by real-time polymerase chain reaction. Cell migration and invasion were assessed using Boyden chamber and invasion assays, respectively. The effects of RANKL on signal transduction molecules were determined by western blot analyses.
  
  
  RESULTS
  We found that stimulation by RANKL altered the cell morphology to the mesenchymal phenotype in normal breast epithelial and breast cancer cells. In addition, RANKL increased the expression levels of vimentin, N-cadherin, Snail, and Twist and decreased the expression of E-cadherin. We also found that RANKL activated nuclear factor-κB (NF-κB), but not extracellular signal-regulated kinase 1/2, Akt, mammalian target of rapamycin, c-Jun N-terminal kinase, and signal transducer and activator of transcription 3. Moreover, dimethyl fumarate, a NF-κB inhibitor, inhibited RANKL-induced EMT, cell migration, and invasion, and upregulated the expressions of Snail, Twist, vimentin, and N-cadherin.
  
  
  CONCLUSIONS
  The results indicate that RANKL induces EMT by activating the NF-κB pathway and enhancing Snail and Twist expression. These findings suggest that the RANKL/RANK system promotes tumor cell migration, invasion, and metastasis via the induction of EMT.
authors.affiliation: Division of Pharmacotherapy, Kinki University School of Pharmacy, Kowakae, Higashi-Osaka 577-8502, Japan. nishida@phar.kindai.ac.jp.
authors.forename: Masanobu
authors.initials: M
authors.lastname: Tsubaki
authors.forename: Makiko
authors.initials: M
authors.lastname: Komai
authors.forename: Shin-Ichiro
authors.initials: S
authors.lastname: Fujimoto
authors.forename: Tatsuki
authors.initials: T
authors.lastname: Itoh
authors.forename: Motohiro
authors.initials: M
authors.lastname: Imano
authors.forename: Kotaro
authors.initials: K
authors.lastname: Sakamoto
authors.forename: Hirotaka
authors.initials: H
authors.lastname: Shimaoka
authors.forename: Tomoya
authors.initials: T
authors.lastname: Takeda
authors.forename: Naoki
authors.initials: N
authors.lastname: Ogawa
authors.forename: Kenji
authors.initials: K
authors.lastname: Mashimo
authors.forename: Daiichiro
authors.initials: D
authors.lastname: Fujiwara
authors.forename: Junji
authors.initials: J
authors.lastname: Mukai
authors.forename: Katsuhiko
authors.initials: K
authors.lastname: Sakaguchi
authors.forename: Takao
authors.initials: T
authors.lastname: Satou
authors.forename: Shozo
authors.initials: S
authors.lastname: Nishida
mesh_terms: D000818:Animals; D001943:Breast Neoplasms / Q000235:genetics / Q000378:metabolism* / Q000473:pathology*; D045744:Cell Line, Tumor; D002465:Cell Movement / Q000502:physiology; D004195:Disease Models, Animal; D018450:Disease Progression; D058750:Epithelial-Mesenchymal Transition / Q000502:physiology; D005260:Female; D006801:Humans; D061986:MCF-7 Cells; D016328:NF-kappa B / Q000235:genetics / Q000378:metabolism*; D009361:Neoplasm Invasiveness; D009687:Nuclear Proteins / Q000096:biosynthesis*; D053245:RANK Ligand / Q000378:metabolism*; D053246:Receptor Activator of Nuclear Factor-kappa B / Q000378:metabolism*; D015398:Signal Transduction; D000071250:Snail Family Transcription Factors; D014157:Transcription Factors / Q000096:biosynthesis*; D051879:Twist-Related Protein 1 / Q000096:biosynthesis*; D015854:Up-Regulation
chemical_list: D016328:NF-kappa B; D009687:Nuclear Proteins; D053245:RANK Ligand; D053246:Receptor Activator of Nuclear Factor-kappa B; C000604407:SNAI1 protein, human; D000071250:Snail Family Transcription Factors; C505686:TNFRSF11A protein, human; C505688:TNFSF11 protein, human; C103173:TWIST1 protein, human; D014157:Transcription Factors; D051879:Twist-Related Protein 1