A REGULATORY AXIS CONNECTING PKCΑ AND ZEB1 MODULATES EPITHELIAL-MESENCHYMAL TRANSITION AND INVASIVENESS OF BREAST CANCER CELLS

LLORENS, M. C.; ROSSI, F. A.; GARCIA I.A.; COOKE M.; ROSSI M.; BOCCO J.L.; KASANIETZ M.; SORIA G.

Salta

Congreso; Joint LV Annual SAIB Meeting and XIV PABMB Congress; 2019

Sociedad Argentina de Investigación en Bioquímica y Biología Molecular

The Epithelial-Mesenchymal Transition (EMT) is an essential program of embryogenesis and tumor progression, ZEB1 is a master regulator of the EMT. While extensive evidence confirmed the importance of ZEB1 as an EMT transcription factor that promotes tumor invasiveness and metastasis, little is known about its regulation. 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. We screened for potential regulatory links between ZEB1 and multiple cellular kinases. Our preliminary in silico studies revealed a plethora of potential phosphorylation sites for several kinases. Due to this level of complexity, we decided to follow 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 a luciferase reporter assay in cells. 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 and full length ZEB1 (ZEB1-FL) levels were actively reduced when NMuMMG-NZEB1 or MDA-MB-231cells were treated with the pharmacological inhibitors of PKCs GF109203X and Gö69761. To study the penetrance of this phenotype with ZEB1-FL, 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. Subsequent validation experiments using 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 the inhibition of mesenchymal phenotypes, including inhibition in cell migration and invasiveness. These findings were extended to an in vivo model, by demonstrating that the stable knockdown of PKCα using lentiviral shRNAs markedly impaired the metastatic potential of MDA-MB-231 breast cancer cells. 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, which might stimulate the evaluation of PKC inhibitors for metastatic breast cancer therapy.