p63 regulates cell proliferation and cell cycle progression‑associated genes in stromal cells of giant cell tumor of the bone

Abstract

Giant cell tumor of bone (GCT) is a destructive neoplasm of uncertain etiology that affects the epiphyseal ends of long bones in young adults. GCT stromal cells (GCTSCs) are the primary neoplastic cells of this tumor and are the only proliferating cell component in long-term culture, which recruits osteoclast-like giant cells that eventually mediate bone destruction. The oncogenesis of GCT and factors driving the neoplastic stromal cells to proliferate extensively and pause at an early differentiation stage of pre-osteoblast lineage remain unknown. Overexpression of p63 was observed in GCTSCs and there is growing evidence that p63 is involved in oncogenesis through different mechanisms. This study aimed to understand the specific role of p63 in cell proliferation and oncogenesis of GCTSCs. We confirmed p63 expression in the mononuclear cells in GCT by immunohistochemical staining. By real-time PCR analysis, we showed a higher level (>15‑fold) of TAp63 expression in GCTSCs compared to that in mesenchymal stem cells. Furthermore, we observed that knockdown of the p63 gene by siRNA transfection greatly reduced cell proliferation and induced cell cycle arrest at S phase in GCTSCs. We found that the mRNA expression of CDC2 and CDC25C was substantially suppressed by p63 knockdown at 24-72 h. Moreover, p63 was found to be recruited on the regulatory regions of CDC2 and CDC25C, which contain p53-responsive elements. In summary, our data suggest that p63 regulates GCTSC proliferation by binding to the CDC2 and CDC25C p53-REs, which may inhibit the p53 tumor suppressor activity and contribute to GCT tumorigenesis.

Figure 1.

p63 protein and gene expression in GCT. (A and B) Immunostaining for p63 in paraffin-embedded tissue sections of two representative cases of GCT showed expression mainly in mononuclear cells (arrows); scale bar, 50 μm. (C) TAp63 isoform was detected in the cell lines and DNp63 was at undetectable level. (D) GCTSCs overexpressed TAp63 (>15-fold) when compared with MSCs; error bars represent SD of three independent experiments, ^**p<0.001 in Dunnett’s multiple comparison test.

Figure 2.

p63 siRNA abrogates both (A) mRNA and (B) protein expression of p63 in GCTSC. GCTSCs were treated with either control siRNA (control) or p63 siRNA (p63RNAi) from 24–72 h. Gene expression was assessed by real-time PCR, p<0.01 by Student’s t-test and protein level was confirmed by western blot analysis. Expression of β-actin is shown for comparison.

Figure 3.

(A and B) Phrase contrast micrographs showing the morphology of MSCs and (C–F) two different GCTSC cell lines were transfected with either (A, C and E) control siRNA or (B, D and F) p63 siRNA for 72 h. Suppression of endogenous p63 reduced cell density; scale bar represents 100 μm.

Figure 4.

Suppression of p63 expression greatly reduced cell proliferation and induced S-phase arrest in GCTSC. MSCs and GCTSCs were transfected with control siRNA or p63 siRNA, cell proliferation was measured by (A) BrdU incorporation assay after 72 h, ^**p<0.01 compared with control by Student’s t-test. The cell cycle analysis was performed with Modfit 3.0 (Becton-Dickinson). (B) A representative graph is shown from three independent experiments, ^*p<0.05 compared with control by Student’s t-test.

Figure 5.

(A) Suppression of p63 expression reduced CDC2 and CDC25C gene expression. MSCs and GCTSCs were transfected with control or p63 siRNA. The mRNA expression of TAp63, CDC2 and CDC25C was evaluated by real-time PCR. Error bars represent SD of three independent experiments, ^**p<0.01 compared with control by Student’s t-test. CDC2 protein level was also downregulated by p63 siRNA treatment at 48 h in GCTSCs and measured (B) by western blot analysis. (C) In vivo binding of p63 to the regulatory regions of CDC2 and CDC25C genes. Formaldehyde cross-linked chromatin was extracted from two different GCTSC cell lines and immunoprecipitated with the p63 antibody. DNA was extracted and analyzed by PCR. One representative result of three independent experiments is shown.