Phorbol myristate acetate suppresses breast cancer cell growth via down-regulation of P-Rex1 expression

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).


Western blot (WB)
Protein samples extracted from exponentially growing cells were loaded on SDS-PAGE, electrophoresed and transferred to Immobilon-FL membrane (Millipore) as described previously (Wong et al., 2011). Primary antibodies were used to identify the relevant protein of interest and the loading control (β-actin). IRdye700 or IRdye800 labeled secondary antibodies (LI-COR Biosciences) (Lincoln, NE) were used for protein band detection. The images were captured with a LI-COR Odyssey infrared imaging system (LI-COR Biosciences). For the quantification of the WB results, the intensity of each band was divided by the β-actin intensity of that same sample and subsequently divided by the corrected intensity of the untreated sample of the same blot. 3

Expression of recombinant PKC isoforms or P-Rex1
MCF-7 or BT-474 cells (1 x 10 6 ) were electroporated with 1.5 µg of either pcDNA3.1 control vector or vector encoding PKCε WT, PKCε CAT, PKCδ WT, PKCδ CAT, PKCη WT, or PKCη CAT. Cells were seeded on 6-well plates and harvested 72 h later for WB analysis of P-Rex1 expression. To determine the effect of exogenous P-Rex1 on PMA inhibition of breast cancer cell growth, MCF-7 and BT-474 cells were transfected with control vector or vector encoding P-Rex1 48 h prior to PMA (10 ng/ml) treatment and were counted 24 and/or 48 h later.

Cell growth assay
MCF-7 cells transfected with PKC isoforms for 24 h were seeded into 24-well plates at a density of 5×10 4 cells/well in IMEM phenol red-free medium supplemented with 2% fetal bovine serum. Cells were cultured in the presence or absence of 2 µM of the PKC inhibitor Gö6983 for 48 h. Cell number was counted with a hemocytometer (Fisher Scientific). Relative cell growth refers to increased cell number, normalized by cell number before Gö6983 treatment.  Non-radioactive measurement of PKC kinase activity MCF-7 and BT-474 cells were cultured overnight in IMEM medium supplemented with 2% fetal bovine serum and then treated without or with PMA (10 ng/ml) for 60 min. Cells were lysed on ice for 30 min with the lysis buffer (20 mM MOPS, pH 7.4, 150 mM NaCl, 5 mM EGTA, 2 mM EDTA, 1% NP40, 1 mM phenylmethylsulfonyl fluoride, 1 mM sodium orthovanadate, 1 mM dithiothreitol, 1 mM benzamidine and 10 µg/ml of leupeptin and aprotinin). Following sonication (3 × 10 sec pulses), the cell lysates were centrifuged at 13,000 ×g for 15 min and the supernatants were collected for PKC kinase activity assay. 50 µg of cell lysates were loaded in duplicate wells on the PKC kinase activity plate of a PKC kinase activity assay kit (Enzo Life Sciences, Farmingdale, NY). The assay was performed following the manufacturer's instruction. The PKC kinase activity was measured at an absorbance of 450 nm on the Synergy™ HTX Multi-Mode Microplate Reader from BioTek (Winooski, VT). Varying quantities of purified active PKC were also assayed to generate a standard curve.

Immunofluorescence microscopy
Monolayered MCF-7 cells on coverglass slides were cultured overnight in IMEM medium supplemented with 2% fetal bovine serum. Cells were treated with PMA (10 ng/ml) for 30 min and then fixed with 4% paraformaldehyde for 10 min. PKCα and ε isoforms were visualized by anti-PKCα poly-clonal and anti-PKCε mono-clonal antibodies (BD bioscience), followed by Alexa Fluor 488-labeled anti-rabbit IgG or anti-mouse IgG, respectively. Images were captured by using a confocal microscope (Leica TCS SP8 MP) and LAS imaging software.

Statistical analysis
All experiments were repeated 3-5 times in duplicates or triplicates. Results are expressed as the mean ± S.E.. Groups were compared using a Student's t-test for unpaired observations or a 2-way ANOVA with the Bonferroni correction where there were multiple comparisons. A p value <0.05 was considered to be significant.