INTERPLAY BETWEEN ZEB1 AND PKCα IN THE REGULATION OF THE EPITHELIAL-MESENCHYMAL TRANSITION PROGRAM

LLORENS, MARÍA CANDELARIA; VAGLIENTI, VICTORIA; KAZANIETZ, MARCELO; LOPEZ HABER, CYNTHIA; ANDINO, DIEGO; SORIA GASTON; BARRIO-REAL, LAURA; CABANILLAS, ANA MARÍA

San Carlos de Bariloche

Congreso; SISTAM; 2018

The Epithelial-Mesenchymal Transition (EMT) is an essential program of embryogenesis and tumor progression, which is tightly regulated by the transcription factor ZEB1. The aim of this work was to explore the signaling pathways that regulate ZEB1 levels and functionality, and how this regulation impacts on the dynamics of the EMT in cancer cells. Our preliminary in silico studies revealed a plethora of potential phosphorylation sites for several kinases. This led us to generate deletions mutants of ZEB1 to find the smaller variant that recapitulates the EMT phenotype of full length ZEB1 (nZEB1). Intriguingly, we found that nZEB1 is enriched in PKC-specific sites and a substrate of p-PKC antibodies in cell extracts, thus suggesting an unforeseen regulatory role of PKC kinases on ZEB1 biology. Our initial experiments showed that nZEB1 levels are actively reduced when cells are treated with the pharmacological inhibitors of PKCs GF109203X and Gö69761. To study the penetrance of this phenotype with full length ZEB1, we investigated the levels of three well-known PKCs paralogs (α, δ and ε), ZEB1 and EMT makers in a group of 9 breast cancer cell lines. Strikingly, we found that PKCα and ZEB1 had a significant positive correlation, being both proteins overexpressed in cell lines with more aggressive phenotypes. To determine if PKCα inhibition triggers the downregulation of endogenous ZEB1 levels, we performed experiments using siRNAs, shRNAs and pharmacological inhibitors in MDA-MB231 cells. We observed a systematic decrease of ZEB1 levels after PKCα inhibition, which was followed by the increased expression of the EMT marker E-cadherine. In line with this finding, these cells depicted a strong decrease in the migration ability and invasiveness in Matrigel.Conclusion: We demonstrated for the first time that the PKCα signal transduction pathway regulates the biological function of ZEB1, defining a novel regulatory axis of the EMT program in breast cancer cell lines.