title: Ras induces experimental lung metastasis through up-regulation of RbAp46 to suppress RECK promoter activity.
abstract: BACKGROUND
  Mutant Ras plays multiple functions in tumorigenesis including tumor formation and metastasis. Reversion-inducing cysteine-rich protein with Kazal motifs (RECK), a metastasis inhibitor gene, suppresses matrix metalloproteinase (MMP) activity in the metastatic cascade. Clarifying the relationship between Ras and RECK and understanding the underlying molecular mechanism may lead to the development of better treatment for Ras-related tumors.
  
  
  METHODS
  Suppression subtractive hybridization PCR (SSH PCR) was conducted to identify Ha-ras (val12) up-regulated genes in bladder cancer cells. Stable cell lines of human breast cancer (MCF-7-ras) and mouse NIH3T3 fibroblasts (7-4) harboring the inducible Ha-ras (val12) oncogene, which could be induced by isopropylthio-β-D-galactoside (IPTG), were used to clarify the relationship between Ras and the up-regulated genes. Chromatin immunoprecipitation (ChIP) assay, DNA affinity precipitation assay (DAPA) and RECK reporter gene assay were utilized to confirm the complex formation and binding with promoters.
  
  
  RESULTS
  Retinoblastoma binding protein-7 (RbAp46) was identified and confirmed as a Ha-ras (val12) up-regulated gene. RbAp46 could bind with histone deacetylase (HDAC1) and Sp1, followed by binding to RECK promoter at the Sp1 site resulting in repression of RECK expression. High expression of Ras protein accompanied with high RbAp46 and low RECK expression were detected in 75% (3/4) of the clinical bladder cancer tumor tissues compared to the adjacent normal parts. Ras induced RbAp46 expression increases invasion of the bladder cancer T24 cells and MMP-9 activity was increased, which was confirmed by specific lentiviral shRNAs inhibitors against Ras and RbAp46. Similarly, knockdown of RbAp46 expression in the stable NIH3T3 cells "7-4" by shRNA decreased Ras-related lung metastasis using a xenograft nude mice model.
  
  
  CONCLUSIONS
  We confirmed that RbAp46 is a Ha-ras (val12) up-regulated gene and binds with HDAC1 and Sp1. Furthermore, RbAp46 binds to the RECK promoter at the Sp1 site via recruitment by Sp1. RECK is subsequently activated, leading to increased MMP9 activity, which may lead to increased metastasis in vivo. Our findings of Ras upregulation of RbAp46 may lead to revealing a novel mechanism of Ras-related tumor cell metastasis.
authors.affiliation: Department of Microbiology and Immunology, National Cheng Kung University, Tainan, Taiwan. s5889102@gmail.com.|Institute of Basic Medical Sciences, National Cheng Kung University, Tainan, Taiwan. s5889102@gmail.com.
authors.forename: Hsuan-Heng
authors.initials: HH
authors.lastname: Yeh
authors.affiliation: Department of Microbiology and Immunology, National Cheng Kung University, Tainan, Taiwan. hen@ms8.url.com.tw.
authors.forename: Yu-Fen
authors.initials: YF
authors.lastname: Tseng
authors.affiliation: Department of Microbiology and Immunology, National Cheng Kung University, Tainan, Taiwan. yuchiao.katie@gmail.com.
authors.forename: Yu-Chiao
authors.initials: YC
authors.lastname: Hsu
authors.affiliation: Department of Microbiology and Immunology, National Cheng Kung University, Tainan, Taiwan. littleblue3016@yahoo.com.tw.|Institute of Basic Medical Sciences, National Cheng Kung University, Tainan, Taiwan. littleblue3016@yahoo.com.tw.
authors.forename: Sheng-Hui
authors.initials: SH
authors.lastname: Lan
authors.affiliation: Department of Microbiology and Immunology, National Cheng Kung University, Tainan, Taiwan. blacktea98@yahoo.com.tw.|Institute of Basic Medical Sciences, National Cheng Kung University, Tainan, Taiwan. blacktea98@yahoo.com.tw.
authors.forename: Shan-Ying
authors.initials: SY
authors.lastname: Wu
authors.affiliation: Department of Microbiology and Immunology, National Cheng Kung University, Tainan, Taiwan. giriraghavaraju@gmail.com.
authors.forename: Giri
authors.initials: G
authors.lastname: Raghavaraju
authors.affiliation: Department of Microbiology and Immunology, National Cheng Kung University, Tainan, Taiwan. doglying@gmail.com.
authors.forename: Da-En
authors.initials: DE
authors.lastname: Cheng
authors.affiliation: Department of Medical Research, Chiayi Christian Hospital, Chiayi, Taiwan. yingray.lee@gmail.com.
authors.forename: Ying-Ray
authors.initials: YR
authors.lastname: Lee
authors.affiliation: Department of Parasitology, National Cheng Kung University, Tainan, Taiwan. z7608045@email.ncku.edu.tw.
authors.forename: Tsuey-Yu
authors.initials: TY
authors.lastname: Chang
authors.affiliation: Department of Pathology, National Cheng Kung University, Tainan, Taiwan. chownh@mail.ncku.edu.tw.
authors.forename: Nan-Haw
authors.initials: NH
authors.lastname: Chow
authors.affiliation: Center of Infectious Disease and Signaling Research, National Cheng Kung University, Tainan, Taiwan. hung1228@nhri.org.tw.|National Institute of Cancer Research, National Health Research Institutes, Tainan, Taiwan. hung1228@nhri.org.tw.
authors.forename: Wen-Chun
authors.initials: WC
authors.lastname: Hung
authors.affiliation: Department of Microbiology and Immunology, National Cheng Kung University, Tainan, Taiwan. a713@mail.ncku.edu.tw.|Institute of Basic Medical Sciences, National Cheng Kung University, Tainan, Taiwan. a713@mail.ncku.edu.tw.|Center of Infectious Disease and Signaling Research, National Cheng Kung University, Tainan, Taiwan. a713@mail.ncku.edu.tw.
authors.forename: Hsiao-Sheng
authors.initials: HS
authors.lastname: Liu
mesh_terms: D000818:Animals; D005260:Female; D058851:GPI-Linked Proteins / Q000037:antagonists & inhibitors / Q000378:metabolism*; D011905:Genes, ras / Q000502:physiology; D006801:Humans; D008175:Lung Neoplasms / Q000378:metabolism* / Q000473:pathology; D061986:MCF-7 Cells; D051379:Mice; D008819:Mice, Nude; D041681:NIH 3T3 Cells; D011401:Promoter Regions, Genetic / Q000502:physiology; D056524:Retinoblastoma-Binding Protein 7 / Q000096:biosynthesis*; D015854:Up-Regulation / Q000502:physiology; D001749:Urinary Bladder Neoplasms / Q000378:metabolism / Q000473:pathology / Q000556:secondary
chemical_list: D058851:GPI-Linked Proteins; C490746:RBBP7 protein, human; C115460:RECK protein, human; D056524:Retinoblastoma-Binding Protein 7