Cell Anoikis Resistance Mediated By Fasn Promote Os Metastasis Via Activating PERK1/2 Signaling Pathway

Background:Osteosarcoma is the most common primary malignant bone tumour,predominantly affecting children and adolescents.The pulmonary metastasis of osteosarcoma(OS)occurs commonly,which resulted from anoikis resistant(AR)of tumor cells as reported by previous studies,but the exact roles of AR in osteosarcoma were not fully studied.Our previous investigations showed fatty acid synthase(FASN)was relating to clinical features of patients with OS.In this study,we aim to explore the functions of FASN in the AR OS cells in vitro and in vivo and study the downstream effectors of FASN.Methods:1.Detect the anoikis resistant abilities between osteosarcoma cell lines(143B?MG-63?Saos-2)and non-tumor cell line(hFOB1.19)by anoikis assay,colony formation and flow cytometry apoptosis assay.2.Explore the role of FASN in anoikis resistance.We used Western-blot?qPCR?ICC to evaluate the expression of FASN and the FASN/pERK1/2/Bcl-xl signaling pathway in AR between osteosarcoma cell line(143B ? MG-63 ? Saos-2)and non-tumor cell line(hFOB1.19).3.Down and up-regulated FASN expression to comfirm the function of FASN and the involved mechanisms.We constructed shRNA lentivirus to down-regulate FASN in FASN-overexpression cell lines,constructed over-expression lentivirus to up-regulate FASN in FASN-lowexpression cell lines to study functions of FASN in AR.We used Western-blot to detect the expression of FASN/pERK1/2/Bcl-xl signaling pathway after down-and up-regulating FASN.Furtherly,we use pERK inhibitor to stop pERK1/2/Bcl-xl signaling pathway to observe the AR ability.4.Established AR cell model to further study FASN promote AR.We use repeated suspended-attached cell culture model to isolate the anoikis resistant cell line 143B-AR.Then we detect the anoikis resistance and the expression of FASN/pERK1/2/Bcl-xl signaling pathway to comfirm the function of FASN in AR.5.Comfirmed FASN enhanced metastasis in vivo.The tumors were orthotopically transplanted into the nude mice,we observe the tumorigenicity and in 143 B and 143 BAR group.Then we injected lentivirus into tumors to observe the the tumorous size,weight and lung metastasis in shR-FASN and control group.Results:1.Osteosarcoma cells exhibit anoikis resistant ability.As compared with non-tumor cell hFOB 1.19,143 B and MG-63 cell show higher survival rate and colony number,but low apoptotic rate in suspended culture.2.The expression FASN was higher in osteosarcoma,and in positively related with AR ability and pERK1/2/Bcl-xl.In 143 B and MG-63 cells,FASN were over-expressed,pERK1/2/Bcl-xl were activiated.Whilest in Saos-2 and hFOB1.19 cells,FASN were low-expressed,pERK1/2/Bcl-xl were suppressed.3.Lost and gain functional experiments revealed FASN promote anoikis resistance.In 143 B and MG-63 cells,lost function of FASN reduced cell survival rate and migration,induced apoptosis,suppressed the pERK1/2/Bcl-xl pathway.In Saos-2 and hFOB1.19 cells,gain function of FASN increased cell survival rate and migration,suppressed apoptosis,activated the pERK1/2/Bcl-xl pathway.Addiationally,blocking pERK1/2 cannot completely reverse the effect of FASN overexpression.4.143-AR cell line was anoikis resistant.143 BAR cell showed higher survival rate,migration abiliby and lower apoptotic rate than 143 B cell in suspension.The FASN/pERK1/2/Bcl-xl pathway was activated.Down-regulation of FASN suppressed anokis resistance in 143B-AR cell.5.FASN promote tumorigenicity and metastasis in vivo.The result showed that anoikis resistant 143B-AR cell formed tumor rapidly.Inhibition of FASN decreased the tumorigenicity and pulmonary metastasis of OS.Conclusion: We showed that anoikis resistant and FASN as two interactional factors facilitated the progress of osteosarcoma.FASN may become the therapeutic target that assisting clinical treatment in future years.