A regulatory axis connecting PKCα and ZEB1 modulates epithelial-mesenchymal transition and invasiveness of breast cancer cells

Abstract

The Epithelial-Mesenchymal Transition (EMT) is an essential program of embryogenesis and tumor progression, ZEB1 is amaster regulator of the EMT. While extensive evidence confirmed the importance of ZEB1 as an EMT transcription factor thatpromotes tumor invasiveness and metastasis, little is known about its regulation. The aim of this work was to explore thesignaling pathways that regulate ZEB1 levels and functionality, and how this regulation impacts on the dynamics of the EMT incancer cells. We screened for potential regulatory links between ZEB1 and multiple cellular kinases. Our preliminary in silicostudies revealed a plethora of potential phosphorylation sites for several kinases. Due to this level of complexity, we decided tofollow up this analysis using ZEB1 deletion mutants (ZD1-HD and NZEB1), these constructs represent 60% and 10% of the full-length protein, respectively, and both retain the capacity to repress the E-cadherin promoter in cells, as determined with aluciferase reporter assay in cells. Intriguingly, we found that NZEB1 is enriched in PKC-specific sites and a substrate of p-PKCantibodies in cell extracts, thus suggesting an unforeseen regulatory role of PKC kinases on ZEB1 biology. Our initialexperiments showed that NZEB1 and full length ZEB1 (ZEB1-FL) levels were actively reduced when NMuMMG-NZEB1 orMDA-MB-231cells were treated with the pharmacological inhibitors of PKCs GF109203X and Gö69761. To study thepenetrance of this phenotype with ZEB1-FL, we investigated the levels of three well-known PKCs paralogs (α, δ and ε), ZEB1and EMT makers in a group of 9 breast cancer cell lines. Strikingly, we found that PKCα and ZEB1 had a significant positivecorrelation, being both proteins overexpressed in cell lines with more aggressive phenotypes. Subsequent validation experimentsusing siRNAs against PKCα in MDA-MB231 cells revealed that its knockdown leads to a concomitant decrease in ZEB1 levels,while ZEB1 knockdown had no impact on PKCα levels. Remarkably, PKCα-mediated downregulation of ZEB1 recapitulates theinhibition of mesenchymal phenotypes, including inhibition in cell migration and invasiveness. These findings were extended toan in vivo model, by demonstrating that the stable knockdown of PKCα using lentiviral shRNAs markedly impaired themetastatic potential of MDA-MB-231 breast cancer cells. Conclusion: We demonstrated for the first time that the PKCα signaltransduction pathway regulates the biological function of ZEB1, defining a novel regulatory axis of the EMT program in breastcancer cell lines, which might stimulate the evaluation of PKC inhibitors for metastatic breast cancer therapy.