Highly-expressed P2X7 receptor promotes growth and metastasis of human HOS/MNNG osteosarcoma cells via PI3K/Akt/GSK3β/β-catenin and mTOR/HIF1α/VEGF signaling

Abstract

The P2X7 receptor, an ATP-gated ion channel, is critical for cancer cell growth, invasiveness, and angiogenesis. Previous studies indicate that P2X7 regulates osteoblast proliferation and osteodeposition and that high P2X7 expression has a pro-growth effect in osteosarcoma. However, how it functions in osteosarcoma cell growth and metastasis is not clear. Thus, we elucidated molecular mechanisms of P2X7-dependent positive regulation of osteosarcoma cell proliferation, invasion, migration, epithelial to mesenchymal transition (EMT), and angiogenesis using in vitro and in vivo models. We confirm that P2X7 is highly-expressed in human osteosarcoma tumor tissues and HOS/MNNG, MG63, U2OS, SW1353 and SAOS-2 cell lines. P2X7 receptor stimulation enhanced HOS/MNNG and SAOS-2 cell proliferation, migration and invasion; but knockdown of P2X7 expression or receptor inhibition had opposite effects. P2X7 positively regulated glycogen content, epithelial to mesenchymal transition and stemness of HOS/MNNG cells. P2X7 activation promoted PI3K/Akt/GSK3β/β-catenin and mTOR/HIF1α/VEGF signaling, thereby mediating pro-tumor effects of osteosarcoma cells. Consistent with data from in vitro experiments, systemic administration of P2X7 agonist induced tumor growth, metastasis and tumor-associated bone destruction in osteosarcoma-bearing nude mice, whereas a P2X7 antagonist reversed these effects. Thus, the P2X7 receptor participates in regulation of osteosarcoma growth and metastasis and we offer evidence that P2X7 may be a promising therapeutic target for treating osteosarcoma.

Keywords: P2X7 receptor; PI3K/Akt signaling pathway; angiogenesis; epithelial to mesenchymal transition; osteosarcoma.

Figure 1

P2X7 is highly expressed in osteosarcoma and increases proliferation of HOS/MNNG and SAOS‐2 cells. (a) Immunohistochemical staining for P2X7 in normal bones (n = 2) and stage IV osteosarcomas (n = 10). Specimens included samples from 7 male and 3 female patients with osteoblastic and chondroblastic osteosarcoma (11–50 years‐of‐age). Normal bone samples were obtained from patients undergoing hip replacement for transcervical fracture. Scale = 3 × 3 × 3 cm³. Quantification of P2X7 (b) mRNA and (c) protein in human osteosarcoma cell lines, MG63, HOS/MNNG, SAOS2, SW1353 and U2OS, and human bone marrow mesenchymal stem cells (h‐BMSC) as control. Relative gene or protein expression was assayed by normalizing with GAPDH. HOS/MNNG and SAOS‐2 cell proliferation after 24, 48, and 72 h of treatment with (d) BzATP (5, 25 or 125 μM), (e) A740003 (5 μM) with or without BzATP (125 μM), and (f) lentiviral vectors with or without BzATP (125 μM) (n = 6). Proliferation rate was evaluated using CCK‐8 assay. Data are means ± SD of 6 or 3 independent experiments. ^*** p < 0.001, ^** p < 0.01, ^* p < 0.05 vs. control. [Color figure can be viewed at wileyonlinelibrary.com]

Figure 2

P2X7 increases migration and invasion of HOS/MNNG and SAOS‐2 cells. Microscopic images of wound healing assay data for HOS/MNNG and SAOS‐2 cells treated with (a) BzATP (5, 25 or 125 μM) or (b) A740003 (5 μM) with or without BzATP (125 μM) for 24 h, or (c) lentiviral vectors with or without BzATP (125 μM) (n = 3). Wound healing percentage was evaluated using Image Pro Plus 6.0 software. Microscopic images and Transwell assay data for invading HOS/MNNG and SAOS‐2 cells (×10⁴) treated with (d) BzATP (5, 25 or 125 μM), (e) A740003 (5 μM) with or without BzATP (125 μM) for 12 h, or (f) lentiviral vectors with or without BzATP (125 μM) (n = 3). Cells that traversed the membrane filter to the lower surface were stained with 0.1% crystal violet and counted using Image Pro Plus 6.0 software. Data are means ± SD of 6 or 3 independent experiments. ^*** p < 0.001, ^** p < 0.01, ^* p < 0.05 vs. control. [Color figure can be viewed at wileyonlinelibrary.com]

Figure 3

P2X7 positively regulates EMT and stemness. HOS/MNNG cells were treated with BzATP (5, 25 or 125 μM), A740003 (5 μM) or with or without BzATP (125 μM) for 24 h, or lentiviral vectors bearing scrambled or P2X7 shRNAs with or without BzATP (125 μM). E‐cadherin, fibronectin, vimentin, and snail protein and mRNA quantified with Western blot or qRT‐PCR, respectively. (a) mRNA and (b) protein of EMT in BzATP‐treated (5, 25 or 125 μM for 24 h) HOS/MNNG cells. (c) mRNA and (d) protein in HOS/MNNG cells treated with BzATP (125 μM) or A740003 (5 μM) or both for 24 h. (e) mRNA and (f) protein of EMT markers in HOS/MNNG cells transfected with scrambled or shP2X7 lentiviral vectors or treated with shP2X7 and BzATP (125 μM). (g) CD133^high cell population and (j) ALDH1 expression in HOS/MNNG cells treated with BzATP (5, 25 and 125 μM) for 24 h. (h) CD133^high cell population and (k) ALDH1 expression in HOS/MNNG cells treated with BzATP (125 μM) or A740003 (5 μM) or both for 24 h. (i) CD133^high cell population and (l) ALDH1 expression in HOS/MNNG cells transfected with scrambled or shP2X7 lentiviral vectors or treated with shP2X7 and BzATP (125 μM). Relative gene or protein expression was assayed by normalizing with GAPDH. CD133‐positive cells were counted using a BD FACS flow cytometer. Data are means ± SD of 3 independent experiments. ^*** p < 0.001, ^** p < 0.01, ^* p < 0.05 vs. control.

Figure 4

PI3K/Akt/GSK3β signaling participates in P2X7‐dependent proliferation, migration, invasion and glycogen accumulation. Western blot of HOS/MNNG cells treated with (a) BzATP (5, 25 or 125 μM), (b) A740003 (5 μM) with or without BzATP (125 μM) for 12 h and (c) lentiviral vectors with or without BzATP (125 μM), and probed with total Akt, phosphorylated (p)‐Akt (Ser473), total GSK3β, p‐GSK3β (Ser9), and GAPDH antibodies. Representative results from 3 independent experiments are shown. (d) CCK‐8 cell proliferation assay, (e) wound healing assay, and (f) Transwell invasion assay in HOS/MNNG treated with BzATP (125 μM), PI3K inhibitor LY294002 (20 μM), or A740003 (5 μM) for 12 h. Glycogen content measured with PAS staining in HOS/MNNG cells treated with (g) BzATP (125 μM) or A740003 (5 μM) for 24 h, or (h) lentiviral vectors with or without BzATP (125 μM). Relative protein expression was assayed by normalizing with GAPDH. Proliferation rate was evaluated using CCK‐8 assay. Wound healing percentage was evaluated using Image Pro Plus 6.0 software. Cells that traversed the membrane filter to the lower surface were stained with 0.1% crystal violet and counted using Image Pro Plus 6.0 software. PAS positive cells were counted using Image Pro Plus 6.0 software. Data are means ± SD of 3 independent experiments. ^*** p < 0.001, ^** p < 0.01, ^* p < 0.05 vs. control. [Color figure can be viewed at wileyonlinelibrary.com]

Figure 5

P2X7 activates Wnt/β‐catenin and mTOR/HIF1α/VEGF signaling. (a) mRNA and (d) protein in BzATP‐treated (5, 25 or 125 μM for 24 h) HOS/MNNG cells. (b) mRNA and (e) protein of β‐catenin and TCF1, and (g) immunofluorescent staining for β‐catenin in HOS/MNNG cells treated with BzATP (125 μM) or A740003 (5 μM) for 24 h. (c) mRNA and (f) protein of β‐catenin and TCF1, and (g) immunofluorescent staining for β‐catenin in HOS/MNNG cells transfected with scrambled or shP2X7 lentiviral vectors or treated with shP2X7 and BzATP (125 μM). Expression of total mTOR, phosphorylated (p)‐mTOR (Ser2448) and HIF‐1α in HOS/MNNG cells treated with (i) BzATP (5, 25 or 125 μM) for 24 h, (j) BzATP (125 μM) or A740003 (5 μM) for 24 h, or (k) lentiviral vectors or treated with shP2X7 and BzATP (125 μM). (l) VEGF protein in HOS/MNNG cells treated with BzATP (5, 25 or 125 μM) or A740003 (5 μM) or both for 24 h, or transfected with lentiviral vectors or treated with shP2X7 and BzATP (125 μM). Relative gene or protein expression was assayed by normalizing with Lamin B1 or β‐actin. In Immunofluorescence, secondary antibodies were FITC or Cy3 labeled, and cell nuclei were counterstained with DAPI. VEGF protein was measured using a commercially available human VEGF ELISA kit. Data are means ± SD of 3 independent experiments. ^*** p < 0.001, ^** p < 0.01, ^* p < 0.05 vs. control. [Color figure can be viewed at wileyonlinelibrary.com]

Figure 6

P2X7 induces growth, metastasis and tumor‐associated bone destruction of HOS/MNNG cells injected in Balb‐c/nude mice. HOS/MNNG cells were resuspended at 1 × 10⁵/10 μl in PBS, and injected into the medullary cavity of the right tibia of mice (n = 24). When tumor volumes reached ~10–100 mm³ (day 10 from cell injection) mice were divided into 4 groups (n = 6 mice/group), and treated with 200 μl (ip) of placebo (PBS + 0.005% DMSO), BzATP (2.5 mg/kg), A740003 (0.025 mg/kg) or BzATP (2.5 mg/kg) + A740003 (0.025 mg/kg) every 2 days for 20 days. (a) Tumor volume measured every 2 days for 20 days. Tumor volume was calculated using the following formula: volume = π/6 (w1 × (w2)²), where w1 = major diameter (mm) and w2 = minor diameter (mm) of the tumor. (b) Immunohistochemical staining for Ki67 in tumor tissue sections from osteosarcoma‐bearing mice. (c) PAS staining of tumor tissue for glycogen stores. Ki67 and PAS positive cells were counted using Image Pro Plus 6.0 software. (d) Left: Representative images of gross and H&E staining of lung tissue sections from osteosarcoma‐bearing mice treated with placebo, BzATP, A740003 or BzATP+A740003. Right: Numbers of metastatic nodules in mice of each treatment groups. (e) (left) Representative μCT images and (right) relative volume (BV/TV) of right tibia from osteosarcoma‐bearing mice treated with placebo, BzATP, A740003 or BzATP+A740003. BV/TV bone volume/total volume. Data are means ± SD, n = 6 mice/group. ^*** p < 0.001, ^** p < 0.01, ^* p < 0.05 vs. control. [Color figure can be viewed at wileyonlinelibrary.com]