Phorbol Myristate Acetate Suppresses Breast Cancer Cell Growth via Down‐regulation of P‐Rex1 Expression

Abstract

Phosphatidylinositol‐3,4,5‐trisphosphate‐dependent Rac exchange factor 1 (P‐Rex1) is highly overexpressed in estrogen receptor‐positive and ErbB2‐overexpressing human luminal breast tumors. The present study was to investigate the role and mechanism underlying phorbol‐12‐myristate‐13‐acetate (PMA), a protein kinase C (PKC) activator, down‐regulation of P‐Rex1 in breast cancer cells. P‐Rex1 mRNA and protein expression were determined by real‐time PCR and western blot analysis, respectively. Cell growth was analyzed by counting cells number and 5‐bromo‐2′‐deoxyuridine (BrdU) cell proliferation assay. The translocation of PKC was examined by immunofluorescence staining. Our results showed that treatment with PMA caused a time‐ and concentration‐dependent decrease in P‐Rex1 protein levels in breast cancer MCF‐7 and BT‐474 cells with a maximum reduction of 87.2 ± 1.1 and 57.0 ± 8.6%, respectively, at a concentration of 10 ng/ml. Interestingly, either PMA treatment or silence of P‐Rex1 by siRNA inhibits MCF‐7 and BT‐474 cell growth by 60–80%, which can be attenuated by restoring P‐Rex1 expression. MCF‐7 and BT‐474 cells express PMA‐sensitive conventional (α and β) and novel (δ, ɛ and η) PKC isoforms. Immunofluorescence staining analysis of breast cancer cells showed translocation of PKCs from cytosol to the plasma membrane and/or nucleus, an indication of their activation upon PMA stimulation. The PKC general inhibitor Gö6983, but not the conventional PKC isoform inhibitor Gö6976, blocked PMA inhibition of P‐Rex1 expression and breast cancer cell growth. Expression of a constitutively active PKCɛ mutant suppressed P‐Rex1 expression and breast cancer cell growth, which was blocked by Gö6983 treatment. In contrast, expression of wild‐type PKCɛ, wild‐type or constitutively active PKCδ and PKCη had no effect. Together, our data suggest that PMA suppresses breast cancer cell growth via P‐Rex1 down‐regulation and PKCɛ activation plays a key role in this regulation. Our study may provide a novel strategy for development of chemotherapeutic agents for P‐Rex1‐overexpressing breast cancer patients that develop resistance to anti‐estrogen and/or anti‐ErbB2 therapies.Support or Funding InformationSupported by grants from NIH (5P20GM103489 and R01HL116849), Nebraska State LB595 research program and the National Natural Science Foundation of China (31100595 and 31300683).