Thrombospondin-1 promotes cell migration, invasion and lung metastasis of osteosarcoma through FAK dependent pathway

Abstract

Microenvironment at the metastatic locus usually differs greatly from that present in the site of primary tumor formation and it has a significant impact on the fate of the extravasated cancer cells. We compared gene expression signatures of primary tumors and lung metastatic tumors, and identified Thrombospondin-1 (TSP1) as highly up-regulated in the lung metastatic tumors. Immunohistochemical staining further indicated that TSP1 protein expression was higher in lung metastatic tumors compared to primary tumors in both osteosarcoma xenograft model and human clinical samples. TSP1 mRNA level is significantly associated with the Enneking stage of osteosarcoma and lung metastasis. TGF-β pathways could stimulate the TSP1 expression in osteosarcoma cells. Knockdown of TSP1 expression in osteosarcoma cells dramatically suppressed cell wound healing, migration and invasion. Treatment with recombinant TSP1 protein in osteosarcoma cells significantly promoted cell wound healing, migration and invasion. Meanwhile, suppression of TSP1 in osteosarcoma cells resulted in decreased pulmonary metastasis in vivo. Mechanistically, TSP1 increased expression of metastasis related genes, including MMP2, MMP9 and Fibronectin 1. TSP1 promoted osteosarcoma cell motility through the activation of FAK pathway. Taken together, our study provides evidence of the contributions of TSP1 to the lung metastasis of osteosarcoma and suggests that this protein may represent a potential therapeutic target for osteosarcoma lung metastasis.

Keywords: FAK; lung metastasis; osoteosarcoma; thrombospondin-1.

Figure 1. Expression of TSP1 in osteosarcoma tissues

(A) Primary tumor cells and lung metastatic tumor cells freshly isolated from Well5-derived orthotopic lung metastasis model and its RNA was extracted and analyzed by microarray. Upper panel, the differentially expressed angiogenesis-related genes were selected for heatmap display. Bottom panel, Real-time quantitative PCR was performed to detect TSP1 expression in the expression profile. **P<0.01. (B) Immunohistochemical staining of TSP1 protein level in primary tumor and lung metastasis from Well5 and another osteosarcoma cell line 143B derived orthotopic lung metastasis model. (C) Immunohistochemical staining of TSP1 in primary and metastatic tumor samples of three osteosarcoma patients.

Figure 2. Regulation of TSP1 expression in osteosarcoma cells by TGF-β pathways

(A) Western blotting assays for the levels of TSP1 in Well5 cells treated with PBS, FGF-2(10 ng/ml), EGF (20 ng/ml), VEGF (5 ng/ml), TGF-β1(5 ng/ml) for 12h. GAPDH was used as a loading control. (B) Western blotting assays for the levels of TSP1 in Well5, U2OS, MG63and 143B cells treated with 5 ng/ml TGF-β1 for the indicated time points. GAPDH was used as a loading control.

Figure 3. Effects of TSP1 on wound healing in osteosarcoma cells

(A) The expression of TSP1 in Well5 and U2OS cell lines transfected with negative control (NC) or lenti-shTSP1 was evaluated by qRT-PCR. Data from 3 independent experiments were expressed as the mean ± S.D. **P<0.01. (B) Decreased protein level of TSP1 was revealed by western blotting after transfection with lenti-shTSP1, consistent with that of qRT-PCR. GAPDH was used as a loading control. (C) Wound healing assay using lenti-TSP1 and negative control transfected Well5 and U2OS cells. (D) Wound healing assay using Well5 and U2OS cells treated with PBS (control) or TSP1 2 ug/ml. Microscopic observations were recorded 0, 24 and 48 hours after scratching the cell surface. A representative image from every independent experiment is shown. (E, F) The distances between wound edges of osteosarcoma cells at 0, 24 and 48 hours. ***P<0.001

Figure 4. Effects of TSP1 on migration, invasion ability in osteosarcoma cells

(A, C) Trans-well migration and invasion assay using lenti-TSP1 and negative control transfected Well5 and U2OS cells. (B, D) Trans-well migration and invasion assay using Well5 and U2OS cells treated with PBS (control) or TSP1 2 ug/ml. (E, F) Bar graph of trans-well migration and invasion assay representing the mean value ± SD from independent experiments performed in triplicate. *P < 0.05; **P < 0.01. (G) Western blotting analysis of MMP2, MMP9 and FN1 expression in lenti-TSP1 and negative control transfected Well5 and U2OS cells. (H) Western blotting analysis of MMP2, MMP9 and FN1expression in Well5 and U2OS cells treated with TSP1 2 ug/ml for different time point.

Figure 5. TSP1 promotes osteosarcoma cell migration and invasion through the activation of FAK pathway

(A) Western blotting analysis of ERK, P38MAPK, FAK phosphorylation in lenti-TSP1 and negative control transfected Well5 and U2OS cells. (B) Western blotting analysis of FAK phosphorylation in in Well5 and U2OS cells treated with TSP1 2ug/ml for different time point. (C) Knockdown efficiency of FAK in Well5 cells and U2OS cells as demonstrated by qRT-PCR. **P < 0.01. (D). Western blot detected the MMP2, MMP9 and FN1 expression in Well5 and U2OS cells after FAK were silenced by siRNAs. (E-H) Trans-well migration and invasion assay in lenti-shFAK and negative control transfected Well5 and U2OS cells treated with PBS or TSP1 2 ug/ml. Bar graph representing the mean value ± SD from independent experiments performed in triplicate. *P < 0.05; **P < 0.01; ***P < 0.001.

Figure 6. Sliencing of TSP1 inhibits lung metastasis of osteosarcoma cells

(A) Knockdown of TSP1 did not inhibit primary tumor growth of osteosarcoma in vivo (P>0.05). (B) Two representative tissue samples retrieved from primary tumors formed by negative control and lenti-shTSP1 transfected Well5 cells in xenograft, respectively. (C) Macroscopic appearances of lung metastasis are shown. The black arrows indicate macroscopic pulmonary metastatic lesions. (D) Representative hematoxylin-eosin stained images (left panel) and IHC staining images (middle and right panel) of the lungs shown in BC (E) Number of tumor nodules was evaluated and analyzed between Well5 cells transfected with lenti-shTSP1 and negative control group. ***P< 0.001. (F) Mean weight of the lungs. *P< 0.05. All values in D and E are mean+ SD of the 12 mice in negative control group and shTSP1 group, respectively. (G) TSP1 silencing led to later death of mice seen by survival curve analysis. *P< 0.05, n= 12.