title: A role for WISP2 in colorectal cancer cell invasion and motility.
abstract: BACKGROUND
  WNT inducible secreted protein 2 (WISP2) has been linked with a variety of human cancer types and may contribute to cancer metastasis. The current study investigated the importance of WISP2 in colorectal cancer cells, examining the impact of targeting WISP2 on Caco-2 cell invasion and motility together with potential mechanisms of action.
  
  
  METHODS
  WISP2 expression was targeted in Caco-2 cells using a ribozyme transgene system and successful knockdown was verified using reverse transcription-polymerase chain reaction (RT-PCR). The impact of WISP2 knockout (Caco-2(WISP2 KO)) on cell growth, adhesion, motility and invasion was examined using a number of in vitro functional assays. In vitro invasion assays were repeated in the presence of wingless-type MMTV integration site family (WNT) inhibitors (FH535 and IWP-2) to investigate the role of the WNT-signalling pathway in the regulation of cell invasion by WISP2. Quantitative-PCR was conducted to measure matrix metalloproteinase (MMP) expression in control [wild-type (Caco-2(WT)) and cells containing the empty pEF6 plasmid (Caco-2(pEF6))] and Caco-2(WISP2 KO) cells.
  
  
  RESULTS
  WISP2 knockout resulted in a significant increase in Caco-2 cell invasion and motility (p<0.05 in comparison to wild-type and plasmid control Caco-2 cells). WISP2 knockout had no significant effect on Caco-2 cell growth rate in 3- and 5-day incubation and no significant impact on Caco-2 cell-matrix adhesion rates (p>0.05). Expression analysis of a number of MMPs indicated an insignificant up-regulation of MMP2, MMP9 (p>0.05) but significant up-regulation of MMP7 (p=0.025) in Caco-2(WISP2 KO) cells compared to controls. Inhibition of WNT signalling using FH535 and IWP-2 brought about a significant or borderline significant decrease in Caco-2(WISP2 KO) cell invasion (FH535 p=0.065) and (IWP-2 p=0.002) and negated the pro-invasive effect of targeting WISP2 in Caco-2 cells.
  
  
  CONCLUSIONS
  WISP2 knockout significantly increased Caco-2 cell invasion and motility. Up-regulation of MMP2, -7 and -9 may indicate that WISP2 regulates invasion and motility through MMPs. Regulation of invasion by WISP2 may involve the WNT signalling pathway.
authors.affiliation: Metastasis and Angiogenesis Research Group, Institute of Cancer and Genetics, Cardiff University School of Medicine, Heath Park, Cardiff CF14 4XN, UK.
authors.forename: Kathryn A
authors.initials: KA
authors.lastname: Frewer
authors.forename: Andrew J
authors.initials: AJ
authors.lastname: Sanders
authors.forename: Sioned
authors.initials: S
authors.lastname: Owen
authors.forename: Natasha C
authors.initials: NC
authors.lastname: Frewer
authors.forename: Rachel
authors.initials: R
authors.lastname: Hargest
authors.forename: Wen G
authors.initials: WG
authors.lastname: Jiang
mesh_terms: D055511:CCN Intercellular Signaling Proteins / Q000235:genetics*; D018938:Caco-2 Cells; D045744:Cell Line, Tumor; D002465:Cell Movement / Q000235:genetics; D015179:Colorectal Neoplasms / Q000235:genetics* / Q000473:pathology; D015972:Gene Expression Regulation, Neoplastic; D006801:Humans; D020782:Matrix Metalloproteinases / Q000096:biosynthesis; D009361:Neoplasm Invasiveness / Q000235:genetics*; D012097:Repressor Proteins / Q000235:genetics*; D060449:Wnt Signaling Pathway / Q000235:genetics*
chemical_list: D055511:CCN Intercellular Signaling Proteins; C413323:CCN5 protein, human; D012097:Repressor Proteins; D020782:Matrix Metalloproteinases
keywords: WISP2; cell invasion; cell motility; colorectal cancer