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

Elpetra Timmermans-Sprang,Meike Philipsen,Jan A Mol,Rob Collin,Arjen Henkes

doi:10.18632/oncotarget.26873

Elpetra Timmermans-Sprang, Meike Philipsen + Show 3 more

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https://doi.org/10.18632/oncotarget.26873

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Abstract

ABSTRACTPurpose: 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 ß-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.

Highlights

Breast cancer is one of the most frequently occurring cancers in the world and, with an estimated 1.7 million new cases diagnosed worldwide each year, it remains a global health challenge
winglesstype MMTV integration site family (Wnt) promotes the outgrowth of metastatic lesions and cancer stem cells (CSC), breast CSCs have an active Wnt signaling and more than half of the breast cancers have an activated canonical Wnt signaling which is associated with a lower overall survival [13,14,15,16]
In recent years we investigated the role of Wnt signaling in canine mammary cancer and found high basal, ligand-independent Wnt activity in a subset of canine mammary tumor cell lines [20]

Summary

Introduction

Breast cancer is one of the most frequently occurring cancers in the world and, with an estimated 1.7 million new cases diagnosed worldwide each year, it remains a global health challenge. Cancer stem cells (CSCs) are thought to play an essential role in enhanced self-renewal ability, therapeutic resistance and metastasis [5, 6]. Wnt promotes the outgrowth of metastatic lesions and CSCs, breast CSCs have an active Wnt signaling and more than half of the breast cancers have an activated canonical Wnt signaling which is associated with a lower overall survival [13,14,15,16]. In both human and canine mammary cancer the enhanced canonical Wnt activity can not be explained by mutations that cause Wnt activation in for instance colon cancer [15, 17]

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