Figure 1

RUNX2 has an anti-mitogenic function in MDA-MB-231 cells. (A) Western blot analysis shows that RUNX2 protein expression is evident in MDA-MB-231 cells but below the level of detection in MCF7 cells. (B) Serum stimulation of MDA-MB-231 cells enhances cell proliferation as reflected by cell counts of cultures supplemented with and without serum (on Days 0 and 3). Cells were plated, serum starved for 48 h and then released for 24 h with and without 10% serum. Live cell counts were performed in triplicate using trypan blue dye-exclusion. (C) Serum starvation of MDA-MB-231 cells increases RUNX2 and p21 protein levels. MDA-MB-231 cells were supplemented with (lane 1) and without (lane 2) serum. The protein expression levels of RUNX2, p21, ERK1/2 and phospho-ERK were analyzed by western blotting. ERK1/2 antibody was used to detect the endogenous level of total-ERK protein in the cells. GAPDH provided a control for equal loading of lysates. (D) The MEK inhibitor PD98059 and the epidermal growth factor receptor (EGFR) kinase inhibitor each significantly increase the expression of RUNX2 in MDA-MB-231 cells by western blotting; β-actin was used as a control for protein loading. The phosphoinositide 3-kinase (PI3K) inhibitor Wortmannin has no effects on RUNX2 levels. MDA-MB-231 cells were incubated in complete medium for 2 h with each of the inhibitors. (E) RUNX2 remains stable for 4 h after serum re-activation. MDA-MB-231 cells were serum starved overnight and serum stimulated for the indicated lengths of time (in hours). Cells were also transfected in parallel with an empty vector (odd lanes) or a viral vector expressing shRUNX2 (even lanes) that 'knock down' (kd) RUNX2 levels and confirms specificity of the RUNX2 signal in MDA-MB-231 cells. (F) Western blot analyses of MDA-MB-231 cells that were first serum starved for 48 h and then treated with either 10% serum or 10% serum in the presence of the protein synthesis inhibitor cycloheximide (100 μM) for the indicated time periods (in hours). RUNX2 blots are represented as short (SE) or long (LE) exposures. Cycloheximide treatment results in a consistent decrease in RUNX2 levels together with a concomitant decrease in p21 and cyclin D1. Comparing RUNX2 protein levels at 8 h of serum activation with and without cycloheximide treatment reveals that RUNX2 is destabilized within 8 h after serum stimulation. These findings suggest that RUNX2 levels are tightly regulated by protein degradation during the cell cycle in MDA-MB-231 breast cancer cells as is the case in normal osteoblastic cells. Thus, elevated expression of RUNX2 protein in MDA-MB-231 breast cancer cells does not appear to be due to abrogation of a protein destabilizing mechanism.