Aberrant Wnt/β-catenin signaling and elevated expression of stem cell proteins are associated with osteosarcoma side population cells of high tumorigenicity

Abstract

According to the cancer stem cell theory, the presence of a small sub‑population of cancer cells, termed cancer stem cells (CSCs), have a significant implication on cancer treatment and are responsible for tumor recurrence. Previous studies have reported that alterations in the Wnt/β‑catenin signaling are crucial in the maintenance of CSCs. In the present study, the characteristic features and activation of Wnt/β‑catenin signaling in CSCs from osteosarcoma, an aggressive human bone tumor, were investigated. In total, ~2.1% of the cancer stem‑like side population (SP) cells were identified in the osteosarcoma samples. The results of subsequent western blot and reverse transcription‑quantitative polymerase chain reaction analyses revealed that the protein levels of β‑catenin and cyclin D1 were markedly upregulated in the fluorescence‑activated cell sorted osteosarcoma SP cells. In addition, the elevated expression levels of stem cell proteins, including CD133, nestin Oct‑4, Sox‑2 and Nanog were significantly higher in the SP cells, which contributed to self‑renewal and enhanced the proliferation rate of the SP cells. Furthermore, the SP cells were found to be highly invasive and able to form tumors in vivo. Taken together, these data suggested that the identification of novel anticancer drugs, which suppress the Wnt/β‑catenin signaling and its downstream pathway may assist in eradicating osteosarcoma stem cells.

Figure 1

Dot-plot analysis of FACS for sorting of osteosarcoma SP cells. (A) Cells were stained with Hoechst 33342 dye and SP cells of 2.1% were identified and outlined (gated population). (B) Percentage of SP cells was significantly reduced to 0.3% in the presence of verapamil. (C) Graphical representation of the SP cells with and without verapamil treatment. This quantitative graph was constructed with data from the dot blot analysis using FACS. SP, side population; FACS, fluorescence-activated cell sorting.

Figure 2

Phenotypic characterization of FACS-sorted osteosarcoma SP cells. (A) In vitro cell proliferation assay. The cell proliferation rates of the SP cells were significantly higher than those of the non-SP cells. The x-axis represents days (D) 1–7, the y-axis indicates the corresponding OD value at 450 nm. (B) Comparison of cell survival rates of the SP cells and non-SP cells following treatment with etoposide, gemcitabine, 5-FU, cisplatin, paclitaxel and oxaliplatin. The SP cells exhibited increased resistance and increased survival rates (>80%), compared with the non-SP cells (<35%). (C) Immunocytochemistry analysis of the sorted SP cells (magnification, x100). SP cells exhibited enhanced expression of ABCG2 (stained red), compared with the non-SP cells. Cells were counterstained with hematoxylin. The data are expressed as the mean ± standard deviation. ^*P<0.05; ^**P<0.01 vs. non-SP cells. SP, side population; 5-FU, 5-fluorouracil; OD, optical density.

Figure 3

Elevated Wnt/β-catenin signaling and stem cell proteins in SP cells. (A) Western blot analysis of protein expression in SP and non-SP cells. Equal quantities of protein were loaded in each lane. (B) Elevated expression levels of the Wnt target gene, CCND1, and stem cell genes, OCT-4, SOX2, nestin, CD133, Nanog SP and ABCG2 were detected using reverse transcription-quantitative polymerase chain reaction. Quantification was performed using data of three independent experiments. GAPDH was used as a housekeeping gene. Data are expressed as the mean ± standard deviation. ^**P<0.01 vs. non-SP cells. SP, side population.

Figure 4

Clone formation efficiency of osteosarcoma SP cells. (A) Total number of sarcospheres generated by osteosarcoma SP cells was significantly higher than those generated by the non-SP cells. (B) Representative images of monoclonal spheres formed by osteosarcoma SP cells (magnification, ×100). The sarcospheres were generated rapidly on day 3 (D3) and the size of the spheres were increased on day 6 (D6). (C) SP cell invasiveness was measured using a Matrigel assay. SP and non-SP cells (4×10⁵) were seeded and incubated for 72 h. The graph represents the number of cells, which invaded across the membrane. The data are expressed as the mean ± standard deviation. ^**P<0.01; ^*P<0.05 vs. non-SP cells. SP, side population.

Figure. 5

Tumorigenic potential of osteosarcoma SP cells. Gross tumor images of the tumors derived after 27 days of subcutaneously injected SP and non-SP cells into NOD/SCID mice.