title: OVOL2, an Inhibitor of WNT Signaling, Reduces Invasive Activities of Human and Mouse Cancer Cells and Is Down-regulated in Human Colorectal Tumors.
abstract: OBJECTIVE
  Activation of WNT signaling promotes the invasive activities of several types of cancer cells, but it is not clear if it regulates the same processes in colorectal cancer (CRC) cells, or what mechanisms are involved. We studied the expression and function of OVOL2, a member of the Ovo family of conserved zinc-finger transcription factors regulated by the WNT signaling pathway, in intestinal tumors of mice and human beings.
  
  
  METHODS
  We analyzed the expression of OVOL2 protein and messenger RNA in CRC cell lines and tissue arrays, as well as CRC samples from patients who underwent surgery at Xiamen University in China from 2009 to 2012; clinical information also was collected. CRC cell lines (SW620) were infected with lentivirus expressing OVOL2, analyzed in migration and invasion assays, and injected into nude mice to assess tumor growth and metastasis. Tandem affinity purification was used to purify the OVOL2-containing complex from CRC cells; the complex was analyzed by liquid chromatography, tandem mass spectrometry, and immunoprecipitation experiments. Gene promoter activities were measured in luciferase reporter assays. We analyzed mice with an intestine-specific disruption of Ovol2 (Ovol2(flox/+) transgenic mice), as well as Apc(min/+) mice; these mice were crossed and analyzed.
  
  
  RESULTS
  Analysis of data from patients indicated that the levels of OVOL2 messenger RNA were significantly lower in colon carcinomas than adenomas, and decreased significantly as carcinomas progressed from grades 2 to 4. Immunohistochemical analysis of a tissue array of 275 CRC samples showed a negative association between tumor stage and OVOL2 level. Overexpression of OVOL2 in SW620 cells decreased their migration and invasion, reduced markers of the epithelial-to-mesenchymal transition, and suppressed their metastasis as xenograft tumors in nude mice; knockdown of OVOL2 caused LS174T cells to transition from epithelial to mesenchymal phenotypes. OVOL2 bound T-cell factor (TCF)4 and β-catenin, facilitating recruitment of histone deacetylase 1 to the TCF4-β-catenin complex; this inhibited expression of epithelial-to-mesenchymal transition-related genes regulated by WNT, such as SLUG, in CRC cell lines. OVOL2 was a downstream target of WNT signaling in LS174T and SW480 cells. The OVOL2 promoter was hypermethylated in late-stage CRC specimens from patients and in SW620 cells; hypermethylation resulted in OVOL2 down-regulation and an inability to inhibit WNT signaling. Disruption of Ovol2 in Apc(min/+) mice increased WNT activity in intestinal tissues and the formation of invasive intestinal tumors.
  
  
  CONCLUSIONS
  OVOL2 is a colorectal tumor suppressor that blocks WNT signaling by facilitating the recruitment of histone deacetylase 1 to the TCF4-β-catenin complex. Strategies to increase levels of OVOL2 might be developed to reduce colorectal tumor progression and metastasis.
authors.affiliation: State Key Laboratory of Cellular Stress Biology, Innovation Center for Cell Biology Research, School of Life Sciences, Xiamen University, Xiamen, China.
authors.forename: Guo-Dong
authors.initials: GD
authors.lastname: Ye
authors.affiliation: State Key Laboratory of Cellular Stress Biology, Innovation Center for Cell Biology Research, School of Life Sciences, Xiamen University, Xiamen, China.
authors.forename: Guang-Bin
authors.initials: GB
authors.lastname: Sun
authors.affiliation: State Key Laboratory of Cellular Stress Biology, Innovation Center for Cell Biology Research, School of Life Sciences, Xiamen University, Xiamen, China.
authors.forename: Peng
authors.initials: P
authors.lastname: Jiao
authors.affiliation: State Key Laboratory of Cellular Stress Biology, Innovation Center for Cell Biology Research, School of Life Sciences, Xiamen University, Xiamen, China.
authors.forename: Chen
authors.initials: C
authors.lastname: Chen
authors.affiliation: State Key Laboratory of Cellular Stress Biology, Innovation Center for Cell Biology Research, School of Life Sciences, Xiamen University, Xiamen, China.
authors.forename: Qing-Feng
authors.initials: QF
authors.lastname: Liu
authors.affiliation: State Key Laboratory of Cellular Stress Biology, Innovation Center for Cell Biology Research, School of Life Sciences, Xiamen University, Xiamen, China.
authors.forename: Xiao-Li
authors.initials: XL
authors.lastname: Huang
authors.affiliation: State Key Laboratory of Cellular Stress Biology, Innovation Center for Cell Biology Research, School of Life Sciences, Xiamen University, Xiamen, China; Department of Biological Sciences, National University of Singapore, Singapore, Republic of Singapore.
authors.forename: Rui
authors.initials: R
authors.lastname: Zhang
authors.affiliation: State Key Laboratory of Cellular Stress Biology, Innovation Center for Cell Biology Research, School of Life Sciences, Xiamen University, Xiamen, China.
authors.forename: Wang-Yu
authors.initials: WY
authors.lastname: Cai
authors.affiliation: State Key Laboratory of Cellular Stress Biology, Innovation Center for Cell Biology Research, School of Life Sciences, Xiamen University, Xiamen, China.
authors.forename: Sheng-Nan
authors.initials: SN
authors.lastname: Li
authors.affiliation: State Key Laboratory of Cellular Stress Biology, Innovation Center for Cell Biology Research, School of Life Sciences, Xiamen University, Xiamen, China.
authors.forename: Jia-Fa
authors.initials: JF
authors.lastname: Wu
authors.affiliation: State Key Laboratory of Cellular Stress Biology, Innovation Center for Cell Biology Research, School of Life Sciences, Xiamen University, Xiamen, China.
authors.forename: Yun-Jia
authors.initials: YJ
authors.lastname: Liu
authors.affiliation: State Key Laboratory of Cellular Stress Biology, Innovation Center for Cell Biology Research, School of Life Sciences, Xiamen University, Xiamen, China.
authors.forename: Rong-Si
authors.initials: RS
authors.lastname: Wu
authors.affiliation: State Key Laboratory of Cellular Stress Biology, Innovation Center for Cell Biology Research, School of Life Sciences, Xiamen University, Xiamen, China.
authors.forename: Yuan-Yuan
authors.initials: YY
authors.lastname: Xie
authors.affiliation: Department of Medical Biotechnology and Laboratory Science, Chang Gung University, Taoyuan, Taiwan.
authors.forename: Err-Cheng
authors.initials: EC
authors.lastname: Chan
authors.affiliation: Department of Biological Sciences, National University of Singapore, Singapore, Republic of Singapore.
authors.forename: Yih-Cherng
authors.initials: YC
authors.lastname: Liou
authors.affiliation: State Key Laboratory of Cellular Stress Biology, Innovation Center for Cell Biology Research, School of Life Sciences, Xiamen University, Xiamen, China. Electronic address: bali@xmu.edu.cn.
authors.forename: Bo-An
authors.initials: BA
authors.lastname: Li
mesh_terms: D000818:Animals; D051778:Basic Helix-Loop-Helix Leucine Zipper Transcription Factors / Q000378:metabolism; D018938:Caco-2 Cells; D002465:Cell Movement; D049109:Cell Proliferation; D015179:Colorectal Neoplasms / Q000235:genetics / Q000378:metabolism* / Q000401:mortality / Q000473:pathology / Q000601:surgery; D015536:Down-Regulation; D058750:Epithelial-Mesenchymal Transition; D015972:Gene Expression Regulation, Neoplastic; D005838:Genotype; D045325:HCT116 Cells; D057809:HEK293 Cells; D056284:Histone Deacetylase 1 / Q000378:metabolism; D006801:Humans; D053208:Kaplan-Meier Estimate; D051379:Mice; D008810:Mice, Inbred C57BL; D008819:Mice, Nude; D008822:Mice, Transgenic; D009361:Neoplasm Invasiveness; D009362:Neoplasm Metastasis; D010641:Phenotype; D011401:Promoter Regions, Genetic; D012333:RNA, Messenger / Q000378:metabolism; D013997:Time Factors; D000073940:Transcription Factor 4; D014157:Transcription Factors / Q000235:genetics / Q000378:metabolism*; D014162:Transfection; D047368:Tumor Burden; D060449:Wnt Signaling Pathway; D051176:beta Catenin / Q000378:metabolism
chemical_list: D051778:Basic Helix-Loop-Helix Leucine Zipper Transcription Factors; C495270:CTNNB1 protein, human; C491431:MOVO protein, mouse; C544941:Ovol2 protein, human; D012333:RNA, Messenger; C532096:TCF4 protein, human; D000073940:Transcription Factor 4; D014157:Transcription Factors; D051176:beta Catenin; C110550:HDAC1 protein, human; D056284:Histone Deacetylase 1
keywords: Colon Cancer; Mouse Model; Signal Transduction; Tumor Suppression