P-cadherin mutations are associated with high basal Wnt activity and stemness in canine mammary tumor cell lines

// Elpetra Timmermans-Sprang 1 , Rob Collin 2 , 3 , Arjen Henkes 2 , Meike Philipsen 1 and Jan A. Mol 1 1 Department of Clinical Sciences of Companion Animals, Faculty of Veterinary Medicine, Utrecht University, Utrecht, The Netherlands 2 Department of Human Genetics, Radboud University Medical Center, Nijmegen, The Netherlands 3 Donders Institute for Brain, Cognition and Behaviour, Radboud University Medical Center, Nijmegen, The Netherlands Correspondence to: Jan A. Mol, email: J.A.Mol@uu.nl Keywords: cancer stem cell; canine mammary tumor exome-sequencing; CDH3; Wnt; cSRC Received: October 18, 2018     Accepted: April 04, 2019     Published: April 26, 2019 ABSTRACT Purpose: To find underlying mutations causing highly-activated Wnt activity in mammary tumor cell lines associated with rounded morphology indicative of stemness/EMT. Methods: Stemness of high Wnt cell lines was confirmed using qPCR on selected genes and microRNA profiling, followed by whole-exome sequencing of 3 high Wnt canine mammary tumor cell lines and 5 low/absent Wnt cell lines. Candidate genes were identified and their involvement in Wnt activity investigated using siRNA silencing. Results: The high Wnt cell lines had morphological and gene expression characteristics reminiscent of stemness. All individual cell lines had about 4000 mutations in the exome in comparison to the reference canine genome. The three high basal Wnt cell lines had 167 unique exome mutations. Seven of these mutations resulted in a SIFT score <0.2 of proteins related to Wnt signaling. However, gene silencing did not change the Wnt pathway activation. Renewed analysis with respect to putative relations to Wnt signaling revealed that P-cadherin (CDH3) had three mutations in the coding region of the extracellular domain and was associated with high Wnt signaling. Silencing by siRNA not only in lowered Wnt activity, but also decreased levels of phosphorylated cSRC and sP-cad, and changed cell morphology towards spindle cell appearance. Conclusion: It is concluded that expression of mutated CDH3 is associated with activation of cSRC, stabilization of s-catenin and a rounded morphology related to a stemness/EMT phenotype. A decreased Wnt activity can be found also by cSRC inhibition, but CDH3 silencing has an additional effect on morphology indicating reversal of EMT.