The VTI1A-TCF4 colon cancer fusion protein is a dominant negative regulator of Wnt signaling and is transcriptionally regulated by intestinal homeodomain factor CDX2

<div><p>Sequencing of primary colorectal tumors has identified a gene fusion in approximately 3% of colorectal cancer patients of the <i>VTI1A</i> and <i>TCF7L2</i> genes, encoding a VTI1A-TCF4 fusion protein containing a truncated TCF4. As dysregulation of the Wnt signaling pathway is associated with colorectal cancer development and progression, the functional properties and transcriptional regulation of the VTI1A-TCF4 fusion protein may also play a role in these processes. Functional characteristics of the VTI1A-TCF4 fusion protein in Wnt signaling were analyzed in NCI-H508 and LS174T colon cancer cell lines. The NCI-H508 cell line, containing the <i>VTI1A</i>-<i>TCF7L2</i> fusion gene, showed no active Wnt signaling, and overexpression of the VTI1A-TCF4 fusion protein in LS174T cells along with a Wnt signaling luciferase reporter plasmid showed inhibition of activity. The transcriptional regulation of the <i>VTI1A-TCF4</i> fusion gene was investigated in LS174T cells where the activity of the <i>VTI1A</i> promoter was compared to that of the <i>TCF7L2</i> promoter, and the transcription factor CDX2 was analyzed for gene regulatory activity of the <i>VTI1A</i> promoter through luciferase reporter gene assay using colon cancer cell lines as a model. Transfection of LS174T cells showed that the <i>VTI1A</i> promoter is highly active compared to the <i>TCF7L2</i> promoter, and that CDX2 activates transcription of <i>VTI1A</i>. These results suggest that the VTI1A-TCF4 fusion protein is a dominant negative regulator of the Wnt signaling pathway, and that transcription of <i>VTI1A</i> is activated by CDX2.</p></div

Transcriptional activity of CDX2.

<p><b> a</b> LS174T cells were transfected with the pGL4.10-VTI1A plasmid-735 and co-transfected with a CDX2 expression plasmid. Activity is stated in mean values relative to pGL4.10 activity and corrected with β-galactosidase activity. Error bars indicate SD, **** indicates p<0.0001, n = 4. <b>b</b> The LS174T wild type cell line and a LS174T CDX2 knockout cell line was transfected with the pGL4.10-VTI1A plasmid. Activity is stated in mean values relative to pGL4.10 activity and corrected with β-galactosidase activity. Error bars indicate SD, **** indicates p<0.0001, n = 4.</p

CDX2 regulation of <i>VTI1A</i> transcriptional activity.

<p><b>a</b> LS174T CDX2 ChIP-seq data was used to identify a CDX2 peak in the VTI1A promoter region [<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0200215#pone.0200215.ref030" target="_blank">30</a>], and the JASPAR CORE database identifies two potential binding sites within this region [<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0200215#pone.0200215.ref031" target="_blank">31</a>]. <b>b</b> qPCR was performed on CDX2 immunoprecipitated DNA from LS174T cells with primers amplifying the 100 bp region seen in fig 4A. Error bars indicates SD, ** indicates p<0.01, n = 4 <b>c</b> Plasmids for deletion analysis with different lengths of the VTI1A promoter inserted in the pGl4.10 plasmid. The two possible CDX2 binding sites are marked. The deletion analysis was carried out in LS174T cells and the luciferase activity is stated in mean values relative to the pGL4.10-VTI1A-735 plasmid and is corrected with β-galactosidase activity. Error bars indicate SD, * indicates p<0.05, *** indicates p<0.001, **** indicates p<0.0001, n = 4.</p