| Background:Lung cancer is one of the most malignant tumors with the highest mortality.Worldwide,about 1.2 million people die from lung cancer each year.Among them,80%-85%of lung cancer patients are non-small cell lung cancer(NSCLC).Dysregulation of lipid metabolism,particularly lipogenesis,is involved in the development and progression of lung cancer,but targeting lipid synthesis pathways for cancer therapy is still unmet clinical need.The FDA-approved weight loss drug Orlistat,as a FASN inhibitor,has been shown to inhibit the cell growths of variety of cancers and enhance the effect of chemotherapeutic drugs.But whether Orlistat has any effect in lung cancer cells is rarely reported.In our study,we applied RNA-seq technology to analyze genome-wide gene changes in lung cancer cells treated with Orlistat,and identified FAF2 as a new target.However,the role of FAF2,as a regulator of lipid droplets,in lung cancer has not been reported.Objective:The objectives of this studies were to explore the anticancer activity of Orlistat in lung cancer and systemically analyze the underlying mechanism of action,especially,to verify its effect on the expression of FAF2.In addition,we aim to investigate the prognosis value of FAF2 in NSCLC patients and its role in the proliferation and invasion of NSCLC cells.Methods:Multiple biochemical and biological methods,including MTT assay,clone formation experiments,flow cytometry,Western Blot,lipid probe detection,and animal experiments,were used to study the anticancer activity of orlistat and the underlying mechanism of action in lung cancer cell lines NCI-H1299,A549,and LLC.In addition,FAF2-siRNA or pcDNA-FAF2 were transfected into lung cancer cells to inhibit or overexpress FAF2 in order to determine whether the anticancer activity of Orlistat is mediated by downregulation of FAF2.Moreover,immunohistochemistry,Transwell,and wound healing experiments were used to test whether FAF2 promote the proliferation and invasion of NSCLC cells.TCGA lung cancer database were used to analyze association of FAF2 expression with patients survivals.Results:We found that orlistat inhibited the proliferation and induced ferroptosis-like cell death of lung cancer cells in vitro and inhibited tumor growth in vivo.In addition,orlistat inhibited lipid peroxidization and reduced protein level of glutathione peroxidase 4(GPX4),a ferroptosis regulator.We also identified FAS associated factor2(FAF2)to be a novel orlistat target,which might be partially involved in the anticancer activity mediated by orlistat treatment.Through analyzing The Cancer Genome Atlas(TCGA)lung cancer database,we found that higher expression levels FAF2/UBXD8,a molecule regulating LD formation and homeostasis is associated with shorter survival time of NSCLC patients.Subsequent validation with immunohistochemistry staining of 164 NSCLC tumors and paired normal tissues in tissue microarrays,confirmed that tumor tissues have significantly higher levels of protein expression of FAF2 compared to paired normal tissues(P<0.001),and that patients with higher FAF2 staining scores had overall shorter survival time compared with patients with lower FAF2 staining scores(P<0.05).In addition,knockdown of FAF2 dramatically inhibited proliferation,motility,and invasion of NSCLC cells,suggesting a causal relation between FAF2 expression and cancer cell survival as well as invasiveness.Conclusion:1.Orlistat inhibited the proliferation and induced ferroptosis-like cell death of lung cancer cells in vitro,and induce apoptosis,lipid oxidation,etc.In vivo experiments can delay tumor progression and increase the number of CD45~+leukocytes in TIL.2.Orlistat can down-regulate the expression of FAF2 in lung cancer.3.The expression of FAF2 is negatively correlated with the survival of NSCLC patients.Knockdown of FAF2 dramatically inhibited proliferation,motility,and invasion of NSCLC cells,suggesting a causal relation between FAF2 expression and cancer cell survival as well as invasiveness. |
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