Mechanistic Studies Of Thrombospondin-1-FAK In Promoting Lung Metastasis Of Osteosarcoma

As the tumor microenvironment in metastases is distinct from that in in situ tumors,the metastatic tumor cells are subject to changes in gene expression in order to adapt the new living environment.Those different genes may contribute to the survival of tumor cells in metastatic site.Therefore,in part one we designed a comparison of the mRNA expression profiles of metastatic tumors and orthotopic tumors freshly isolated from the osteosarcoma orthotopic lung metastasis model in mice.Among these different expression genes,the expression of thrombospondin-1(TSP1),an angiogenesis-related gene,was significantly higher in lung metastatic tumors than in primary 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 cell line Well5 and U2 OS dramatically suppressed cell wound healing,migration and invasion.Treatment with recombinant TSP1 protein in these two osteosarcoma cell lines significantly promoted cell wound healing,migration and invasion.Meanwhile,suppression of TSP1 in Well5 cells resulted in decreased pulmonary metastasis in vivo.Mechanistically,TSP1 induced increased expression of metastasis related genes,including MMP2,MMP9 and Fibronectin 1(FN1).And the effects of TSP1 on osteosarcoma cell motility were primarily mediated through FAK dependent 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 the therapeutic target for osteosarcoma lung metastasis.In the second part,we further study the correlation between the expression of FAK and the clinical characteristics of osteosarcoma patients and its role and mechanism in the development of osteosarcoma.In the second part,we further study the correlation between the expression of FAK in the FAK signaling pathway and the clinical characteristics of osteosarcoma patients and its role and mechanism in the development of osteosarcoma.Immunohistochemistry confirmed that p-FAK(Y397)was highly expressed in primary human osteosarcoma tumor samples and was associated with osteosarcoma prognosis and lung metastasis.PF562271,a FAK inhibitor,greatly suppressed the proliferation,colony formation in human osteosarcoma cell lines.In addition,treatment of osteosarcoma cell lines with PF562271 induced apoptosis and down-regulated the activity of AKT/mTOR pathway.Furthermore,PF562271 impaired tube formation ability of endothelial cells in vitro.Finally,oral administration of PF562271 in mice dramatically reduced tumor volume,weight and angiogenesis of osteosarcoma xenografts in vivo.Therefore,FAK plays an important role in the development of osteosarcoma,which is a potential target for osteosarcoma treatment.