Cancer-related ectopic expression of the bone-related transcription factor RUNX2 in non-osseous metastatic tumor cells is linked to cell proliferation and motility

Human MDA-MB-231 and MCF-7 breast cancer cell lines were cultured in Dulbecco's modified Eagle's medium (DMEM, Gibco, Carlsbad, CA, USA) supplemented with 10% fetal bovine serum (FBS, Hyclone, Waltham, MA, USA), 5% L-glutamine (PAA, Pasching, Austria) and 1% penicillin/streptomycin (PAA, Pasching, Austria) at 37°C and 5% CO₂. Cell proliferation was measured by performing live cell counts in triplicate using Trypan Blue exclusion as a measure for cell viability. Inhibition of MAPK dependent signaling pathways was carried out by treatment with the MEK1 inhibitor PD98059 (#9900, Cell Signaling Technology, Inc., Beverly, MA, USA). The inhibitor was prepared as a 10 mM stock solution in dimethyl sulfoxide (DMSO). MDA-MB-231 and MCF-7 cells were plated at a density of 3 × 10⁵ cells per well in six-well plates and incubated overnight. Cells were then treated with various concentrations of PD98059 (that is, 0, 1, 5, 10, 20 and 50 μM) and incubated for two hours at 37°C before preparation of whole cell lysates. Stock cycloheximide was dissolved in DMSO at 100 mM concentration and freshly added into the media.

Lysates were prepared from cells washed with ice-cold phosphate-buffered saline (PBS, pH 7.2), scraped and lysed into 100 μl of 1 × SDS-PAGE protein loading buffer (31.25 mM Tris-HCl, pH 6.8, 12.5% glycerol, 2.5% β-mercaptoethanol, 1% sodium dodecyl sulfate (SDS), 0.005% bromophenol blue and 1% Roche complete protease inhibitors cocktail). The cell suspension was sonicated for a few seconds to disperse the cells and cell debris was pelleted by centrifugation at 14,000 g for 10 minutes at 4°C. The supernatant containing the protein fraction was collected and boiled for five minutes. Protein samples were then stored at -20°C until further analysis.

Equal amounts of protein from each treatment group were resolved on a 10% SDS-PAGE gel (100 V, 120 minutes) and subsequently transferred onto a nitrocellulose membrane (100 V, 60 minutes). Blots were blocked with 5% nonfat dried milk in PBS-0.1% Tween 20 (PBS-T) solution for 30 minutes prior to primary antibody incubation overnight at 4°C. Primary antibodies to proteins of interest were diluted in PBS-T solution containing 3% bovine serum albumin (BSA) at a ratio of 1:1,000. After incubation with primary antibodies, blots were washed three times for five minutes each with PBS-T solution and incubated for one hour at room temperature with the appropriate secondary antibody at a 1:5,000 dilution in PBS-T solution containing 5% nonfat dried milk. Following incubation, blots were washed three times for five minutes each with PBS-T solution, and the antibody binding was detected with SuperSignal West Pico Chemiluminescent substrate (Thermo Scientific, Waltham, MA, USA) by exposing blots to XAR-5 film (Kodak, Rochester, NY, USA).

The primary antibodies used were RUNX2 mouse monoclonal antibody (D130-3, MBL International, Woburn, MA, USA), phospho-p44/42 MAP kinase E10 mouse monoclonal antibody (#9106, Cell Signaling Technology, Inc, Beverly, MA, USA), p44/42 MAP kinase (ERK1/2) rabbit polyclonal antibody (#9102, Cell Signaling Technology, Inc), p21 rabbit polyclonal antibody (sc-397, Santa Cruz Biotechnology, Santa Cruz, CA, USA), p53 rabbit polyclonal antibody (sc-6243, Santa Cruz), c-myc mouse monoclonal antibody (sc-40, Santa Cruz) and GAPDH mouse monoclonal antibody (sc-32233, Santa Cruz). The secondary antibodies used were horseradish peroxidase (HRP)-conjugated goat anti-mouse IgG antibody (sc-2005, Santa Cruz) and HRP-conjugated goat anti-rabbit IgG antibody (sc-2004, Santa Cruz).

Total RNA was prepared according to manufacturer's instructions (Qiagen RNeasy Mini kit, Qiagen, Hilden, Germany). In-column genomic DNA digestions were performed using DNase I (Qiagen). Total RNA collected samples were quantified and reversed-transcribed to cDNA and 5 ng of cDNA was amplified on the ABI 7300 Real-Time PCR System using fluorescent SYBR Green PCR master mix (Fermentas, Thermo Scientific, Waltham, MA, USA) with the following sets of primers: p21 (forward primer: 5'-GTCCGTCAGAACCCATGC-3', reverse primer: 5'-GTCGAAGTTCCATCGCTCA-3'), cyclin D1 (forward primer: 5'-TGAACAAGCTCAAGTGGAACC-3', reverse primer: 5'-GTTTGCGGATGATCTGTTTGT-3') and GAPDH (forward primer: 5'-GAGTCCACTGGCGTCTTCA-3', reverse primer: 5'-GTTCACACCCATGACGAACA-3'). Average fold changes were calculated by differences in threshold cycles (Ct) between pairs of samples. GAPDH gene was used as an internal control.

Wound healing assays were used to measure cell migration. Cells in various groups were seeded in six-well plates at 5 × 10⁵ cells per well to form a confluent layer of cells overnight. A line of cells was mechanically removed by scratching the cell layer with a 200 μl pipette tip to create a 'wound'. Cells migrating into the 'wound' area were monitored at 30 minutes intervals by automated image collection at the same wound location with a Nikon Eclipse Live Cell Imaging system. Three separate regions along the wound were randomly chosen as scratch zones. The data were analyzed using the accompanying Nikon NIS-Elements software provided by the manufacturer (Nikon Corporation, Chiyoda-ku, Tokyo, Japan). During image collection, cells were maintained under sterile culture conditions at 37°C in an atmosphere containing 5% CO₂.

RUNX2 is normally expressed in lineage-committed mesenchymal progenitor cells with a osteogenic cell fate, but RUNX2 expression is also aberrantly induced in different cancer cell types [8‐17]. For example, RUNX2 is endogenously expressed in selected breast cancer cell lines as evidenced by detection of RUNX2 protein by western blot analysis in highly malignant MDA-MB-231 breast adenocarcinoma cells, but not in MCF7 breast adenocarcinoma cells that retain several characteristics of differentiated mammary epithelium (Figure 1A). Comparison of these two cell types may reveal the biological purpose of the aberrant ectopic expression of RUNX2 in non-osteoblastic cancer cells.

In normal osteoblasts, RUNX2 mRNA and protein levels are elevated in quiescent cells upon growth factor deprivation [23, 41], but this growth-related regulation of RUNX2 gene expression is perturbed in several osteosarcoma cell types that express RUNX2 at elevated levels [23‐26]. To assess whether expression of RUNX2 is regulated with respect to cell growth in MDA-MB-231 breast cancer cells, we performed serum starvation and stimulation experiments (Figure 1B). Cells were first cultured overnight in the presence of serum, and then cells were incubated with medium with or without serum for three days. Modest MDA-MB-231 cell proliferation is observed in cells maintained in normal serum (that is, cells increase in number but do not quite double even after three days), but cell proliferation is inhibited in cells subjected to prolonged serum starvation that enter a quiescent state (Figure 1B). Similar to MC3T3 osteoblasts [23, 41], expression of RUNX2 increases with serum starvation and occurs concomitant with loss of phosphorylated Erk (Figure 1C). This finding suggests that RUNX2 expression is inversely correlated with cell proliferation and mitogen-dependent activation of the MEK-Erk pathway.

To examine whether expression of RUNX2 is directly controlled by mitogenic signaling pathways, we subjected proliferating MDA-MB-231 cells to distinct proliferation-associated kinase inhibitors and monitored RUNX2 protein levels by western blotting. MAPK signaling is known to regulate RUNX2 activity in different cell types [53, 54]. The epidermal growth factor (EGF) receptor gene (EGFR/HER1/ERBB1) and other EGF receptors (HER2/NEU/ERBB2, HER3 and HER4) are frequently expressed in human breast cancer cells, including MDA-MB-231 cells [55, 56]. Expression of EGFR and HER2 is associated with aggressive cancers and inhibitors of EGFR-HER signaling are tested in clinical trials [57]. Therefore, it is of interest to investigate links between EGFR signaling and RUNX2 expression. The results clearly show that RUNX2 protein is elevated by inhibition of the Erk pathway using the chemical inhibitor PD98059 or the EGFR pathway using AG494 (Figure 1D). However, inhibition of phosphoinositide 3-kinases (PI3Ks) by Wortmannin has no effect on RUNX2 protein levels (Figure 1D). Hence, inhibition of MAPK and EGFR signaling pathways attenuates RUNX2 levels in MDA-MB-231 cells.

Induction of cell proliferation in quiescent osteoblasts results in down regulation of RUNX2 gene expression at the level of transcription, as well as mRNA and protein accumulation [23, 41]. Therefore, we tested whether serum stimulation of MDA-MB-231 cells can acutely regulate RUNX2 protein levels (Figure 1E). Parallel samples were treated with RUNX2 siRNA which establish the identity of the RUNX2 band in SDS-PAGE. Western blot results clearly show that RUNX2 levels remain high for at least four hours after serum stimulation and perhaps may be modestly and transiently upregulated around 1.5 hours after induction of cell growth as cells presumably exit from quiescence (G0) and enter the G1 phase (Figure 1E). These data suggest that RUNX2 levels are not acutely coupled to mitogenic signaling but may be down-regulated at a later proliferative stage (for example, the G1/S phase transition) that is beyond the four-hours-time point we have examined (Figure 1E).

To assess whether expression of RUNX2 in MDA-MB-231 cells is facilitated by protein stabilization, we reactivated proliferation in serum deprived MDA-MB-231 cells in the absence or presence of the protein synthesis inhibitor cycloheximide (100 μM). Assessment of the natural decay of RUNX2 over an eight-hour time course reveals that RUNX2 levels decrease with a half-life of about four-hours (Figure 1F), which is comparable to the half-life of exogenously expressed RUNX2 in COS cells [47]. For comparison, levels of the labile protein cyclin D1 are rapidly destabilized (less than two hours) albeit that levels rebound as the efficacy of protein inhibition by cycloheximide diminishes over time (Figure 1F). These data suggest that induction of RUNX2 protein expression in MDA-MB-231 cells is not due to abrogation of protein destabilizing mechanisms that normally degrade RUNX2, but may occur via other upstream gene regulatory mechanisms involving, for example, transcription factors or microRNAs.

Because RUNX2 levels in MDA-MB-231 cells increase by serum deprivation and because Erk kinase phosphorylation is a key step in the mitogenic actions of serum-derived growth factors, we assessed whether Erk phosphorylation by MEK is biologically linked to increased RUNX2 protein expression by treating breast cancer cells with the MEK inhibitor PD98059 (Figure 2A, B). We performed western blot analysis of protein lysates from PD98059 treated MDA-MB-231 and MCF-7 cells to assess whether MEK inhibition can modulate RUNX2 expression in cells that ectopically express RUNX2 (that is, MDA-MB-231 cells) or cells in which RUNX2 is below the level of detection (that is, MCF-7 cells). The results show that Erk phosphorylation is decreased in a dose-dependent manner in both cell types (Figure 2). In each case, there is nearly a complete loss of phosphorylated Erk at 10 μM PD98059, albeit that MCF-7 cells appear to be considerably more sensitive to PD98059 inhibition (Figure 2B). Total levels of Erk are not appreciably altered in either MDA-MB-231 or MCF-7 cells at the doses of PD98059 we tested (Figure 2A, B). The concomitant inhibition of cell proliferation that arises from Erk inhibition is reflected by a modest increase in the levels of the CDK inhibitor p21/CDKN1A (Figure 2A, B). Importantly, the levels of RUNX2 are clearly upregulated in MDA-MB-231 cells upon treatment with PD98059 concentrations above 10 μM (Figure 2A), but RUNX2 levels remain below the level of detection in MCF7 cells (Figure 2B). Taken together, these results establish that RUNX2 protein levels are enhanced upon inhibition of mitogenic MEK-Erk signaling in MDA-MB-231 cells, but that this inhibition does not induce RUNX2 in MCF7 cells.

To assess the biological role of RUNX2 in MDA-MB-231 cells, we performed transient knockdown of RUNX2 with siRUNX2. Depletion of RUNX2 decreases the mRNA levels of the cell cycle inhibitor p21 and increases cyclin D1 mRNA levels indicating that loss of RUNX2 provokes a pro-proliferative response (Figure 3A, B). Constitutive knockdown of RUNX2 using a MDA-MB-231 cell line containing a retrovirus that expresses shRUNX2 RNA, also results in reduced p21 mRNA levels (albeit no commensurate change in p21 protein levels) (Figure 3C, D). Levels of p53 remain constant in MDA-MB-231 cells, which express a mutant p53 protein (R175H) which renders p21 gene transcription insensitive to p53. Reduction of RUNX2 has at best a marginal positive effect on cell growth over a period of 10 to 20 days (Figure 3E and data not shown). The rather modest observed variation in cell number at Day 10 may reflect modulations in cell cycle kinetics (duration of progression through each of the cell cycle stages), as well as the balance between cell survival and cell death. However, clear biological differences before Day 10, when shRNA treatments typically have more pronounced direct effects than at later stages, are not evident. Because loss of RUNX2 does not have major proliferative effects, it appears that RUNX2 activity is not critical for growth of MDA-MB-231 cells.

Our preceding results indicate that RUNX2 is a potential cell growth inhibitor thus raising the question why aggressive human derived breast cancer cells would tolerate induction of RUNX2 gene expression. We postulated that RUNX2 may have a pathological side that increases the aggressiveness of MDA-MB-231 cells during metastasis. Therefore, we performed migration assays using a live imaging system and found that loss of RUNX2 by shRNA (Figure 4A) decreases cell motility in wound healing ('scratch') assays (Figure 4B). Time course results revealed that the number of cells migrating into the scratched area was significantly lower at both earlier (4 h) and later (24 h) time-points in cells depleted for RUNX2. We next investigated whether elevated expression of wild type RUNX2 in MCF7 cells, which are breast cancer cells with limited migratory potential and do not express detectable RUNX2 protein, would alter cell migratory potential. Indeed forced expression of RUNX2 increased motility in wound healing assays (Figure 5). Taken together, the results with MDA-MB-231 and MCF7 cells demonstrate that RUNX2 stimulates cell motility.