ZEB1 IS REGULATED BY PKC-ALPHA IN BREAST CANCER CELL LINES

MARÍA CANDELARIA LLORENS DE LOS RÍOS; CYNTHIA LOPEZ HABER; LAURA BARRIO-REAL; MARÍA VICTORIA VAGLIENTI; MARCELO KAZANIETZ; ANA MARÍA CABANILLAS.

Mar del Plata

Congreso; LXI Reunión Científica Anual de la Sociedad Argentina de Investigación Clínica (SAIC).; 2016

Sociedad Argentina de Investigación Clínica

Cancer progression can be activated by cytokine signals which regulate transcription factors such as ZEB1 or Snail. We intended to uncover the role of PKC signaling in ZEB1 regulation. PKC isoforms and EMT markers were determined by immunoblotting in 9 breast cancer cell lines. PKCα and ZEB1 had a significant positive correlation (p≤0.05). The interference of PKCalpha expression by 2 siRNAs significantly reduced ZEB1 expression at 72hs in MDA-MB-231 cells (siPKCα1,2: 0.64±0.06 and 0.67±0.06 vs siNonTarget siNT:1)(p≤0,05) as well as in MDA-MB-453 and BT549 (72/96hs) and in MDA-MB-231 (96hs). EMT markers changed their expression at no time. However, ZEB1 mRNA (qPCR) did not differ from control in siPKCα/MDA-MB231 cells which suggests that PKCα could regulate ZEB1 expression by diminishing its protein stability. In addition, motility and invasive abilities (Matrigel®invasion assay) of MDA-MB-231 cells silenced by 6 siPKCα or 4 siZEB1 lowered significantly against siNT cells (siPKCα1,2,3,4,5,6: 40.3±3.5; 52.6±4.2; 53.4±2.6; 52.0±2.8; 42.7±2.4; 59.6±3.1 vs siNT: 83.1±2.9) (p≤0.0001) (siZEB1 1,2,3,4: 25.2±2.3; 31.0±2.49; 80.3±3.0; 39.7±2.7 vs siNT: 150.6±3.9) (p≤0.0001). We also tested the capacity of actin cytoskeleton to respond to a 10 minute stimulus of 10% Fetal Bovine Serum by Phalloidin-rhodamine stainining on siPKCa or siZEB1MDA-MB-231 cells. Control and siZEB1 cells showed ruffles and lamellipodia structures as a response to 10%FBS while siPKCalpha MDA-MB231 cells were unable to have a normal response. Results were expressed as mean ± SEM. The results suggest that PKCα could regulate ZEB1 expression in breast cancer cell lines by modifying its protein stability at short term. The poor ruffle formation only in siPKCa cells compared to a normal response in siZEB1 cells suggest that PKCalpha is able to regulate cell invasion by two mechanisms: ZEB1 dependent and ZEB1 independent. Conclusion: PKCα is novel regulator of ZEB1 and therefore the cell invasion in breast cancer.