Mechanism Of USP18 On Lung Cancer Metabolism And Metastasis

ObjectivesLung cancers remain leading cause of death worldwide.There is a pressing need to develop to uncover novel molecular targets and effective therapeutics strategy against lung cancer.Recent studies have found that USP18(Ubiquitin-Specific Peptidase 18,USP18)plays a vital role in initiation and progress of lung cancer by stabilizing key oncoproteins including PML/RARα,Kras,Cyclin D and others.We sought to expand our previous study to cancer cellular metabolism and lung cancer metastasis.USP18 knockout mice exhibited lower lipolysis rates,altered fat to body weight ratios and striking cold sensitivity.This indicated USP18 is a regulator of lipid and fatty acid metabolism.We sought to learn if this occurs through altered lipid and fatty acid metabolism.Notably,loss of USP18 repressed adipose triglyceride lipase(ATGL);gain of USP18 expression upregulated ATGL in lung tumors.Immunoprecipitation assays confirmed that ISG15 covalently conjugated to ATGL.Protein expression of thermogenic regulators was examined in brown fat of USP18 null versus wild-type mice.Uncoupling Protein 1(UCP1)was repressed in USP18 null fat.Gain of USP18 expression augmented and stabilized UCP1 protein via reduced ubiquitination.Gain of UCP1 expression independently in human and murine lung cancer cell lines enhanced proliferation.UCP1 knock-down suppressed lung cancer cell proliferation.Gain of UCP1 expression promoted fatty acid beta oxidation.Combined USP18,ATGL and UCP1 profiles were interrogated in The Cancer Genome Atlas(TCGA).Intriguingly,lung cancers with enhanced USP18,ATGL and UCP1 expression had an unfavorable survival.On the other hand,proteomics screening found a metastasis related gene,14-3-3ζ,is stabilized by USP18.Through in vitro and in vivo assays,we established demonstrate that USP18 promote lung cancer metastasis through stabilizing 14-3-3ζ.These findings indicate that USP18 is a molecular pharmacologic target regulating fatty acid metabolism and metastasis.These studies set the stage of USP18 inhibitors development.Methods(1)Relationship between USP18 expression and lung cancer: The Cancer Genome Atlas(TCGA)was analyzed to explore the correlation between USP18 m RNA level and lung cancer patient survival.(2)Loss of USP18 decreases lipolysis: Body composition analysis was performed on USP18 null versus wildtype mice to measure overall lipid metabolism;in vivo lipolysis assay was performed on USP18 null versus wildtype mice for lipolysis rate;Plasma parameter analysis was performed to investigate the effect of USP18 on plasma glucose,total triglycerides,total cholesterol,adiponectin and etc;lipid metabolism analysis was performed to detect the effect of USP18 overexpression on lipid metabolism.(3)The molecular mechanism of USP18 stabilizing ATGL: Western Blotting was performed to learn the deubiquitinase activity of USP18 on ATGL.The interaction between USP18 and ATGL were explored via immunoprecipitation and western blotting.(4)Loss of USP18 result in suppressed lipid metabolism: USP18 null versus wildtype mice were placed in low temperature environment to measure body temperature;western blotting was performed to explore the effect of USP18 expression on UCP1 protein level;seahorse and β-hydroxybutyric acid colorimetric analysis were performed to investigate the effect of UCP1 on fatty acid oxidation rate;Cell Titer-Glo luminescence assay was performed to understand the effect of UCP1 on lung cancer cells proliferation.(5)Augmented USP18,ATGL and UCP1 expression and lung cancer outcome: The Cancer Genome Atlas(TCGA)was analyzed to explore the correlation between the expression level of USP18,ATGL,UCP1 and lung cancer patient survival.(6)The effect of USP18 on lung cancer migration and invasion: Wound healing and transwell assays were performed to explore the effect of USP18 on lung cancer cell metastasis in vitro;syngeneic mouse lung cancer metastasis models were used to explore the effect of USP18 on lung cancer cell metastasis in vivo.(7)The effect of USP18 on lung cancer metastasis by regulating 14-3-3ζ: RPPA was performed to find the metastasis related proteins affected by USP18;western blotting was performed to explore the effect of USP18 on 14-3-3ζ protein levels;transwell assay was performed to investigate the effect of 14-3-3ζ on the lung cancer cells lacking USP18 which have low migration and invasion ability;TCGA database was analyzed to find out the correlation between 14-3-3ζ expression levels and lung cancer patient survival.Results(1)Relationship between USP18 expression and lung cancer: TCGA analysis showed higher USP18 m RNA level in lung cancer tissues than USP18 m RNA in non-cancer lung tissues.(2)Loss of USP18 decreases lipolysis: body composition analysis indicated that fat weight and fat ratios to body weights were significantly increased in USP18 null mice compared to wildtype mice;in vivo lipolysis assays showed that lipolysis rate in USP18 null mice was significantly reduced;plasma parameter analysis indicated that the serum adiponectin and high-density lipoprotein of USP18-deficient mice were significantly reduced.(3)The molecular mechanism of USP18 stabilizing ATGL: knockdown of USP18 can induce ATGL degradation;immunoprecipitation confirmed that ISG15 conjugates with ATGL.(4)Loss of USP18 result in suppressed lipid metabolism: cold exposure assay indicated that the USP18 null mice are more sensitive to cold environment than wildtype mice;depletion of USP18 can induce UCP1 degradation;overexpression of USP18 can upregulate UCP1 protein;Seahorse and β-hydroxybutyric acid colorimetric assays showed that UCP1 overexpression significantly increase fatty acid beta-oxidation;proliferation assay showed that depletion or overexpression of UCP1 decreases or increases lung cancer cell proliferation,respectively.(5)Augmented USP18,ATGL and UCP1 expression and lung cancer outcome: TCGA data revealed that a signature of high USP18-ATGL-UCP1 expression led to a statistically-significant decline in survival as compared to lung cancers having a lower USP18-ATGL-UCP1 expression profile.The same effect was also found in prostatic adenocarcinomas kidney chromophobes and low grade gliomas.(6)The effect of USP18 on lung cancer migration and invasion: migration and invasion assay indicated that depletion of USP18 inhibits lung cancer cell migration and invasion in vitro;mouse syngeneic subcutaneous and tail vein models showed that knockdown of USP18 inhibits lung cancer migration and invasion in vivo.(7)The effect of USP18 on lung cancer metastasis by regulating 14-3-3ζ: RPPA analysis provided positive correlation between 14-3-3ζ and USP18 protein level in human and murine lung cancer cells;western blotting showed that knockdown of USP18 can induce 14-3-3ζ degradation;transwell assay showed that overexpression of 14-3-3ζ rescue the migration and invasion ability of USP18 knockdown lung cancer cells;TCGA analysis showed that of 14-3-3ζ m RNA was not only negatively correlated with the survival of lung cancer patients but also with breast cancer,renal chromosomal cancer,renal papillary cell carcinoma,pancreatic cancer,and uveal melanoma.Conclusion In this study,we found that USP18 plays an important role in lung cancer metabolism and metastasis.USP18 stabilizes of ATGL protein by deconjugating ISG15 from ATGL.ATGL is decreased USP18 null mice,which in turn significantly inhibits lipolysis,increases the fat weight,fat ratio and body weight of mice significantly;loss of USP18 significantly downregulates UCP1 protein,which leads to temperature sensitivity.USP18 promotes fatty acid oxidation through stabilizing UCP1 in lung cancer cells.In the meantime,USP18 can regulate 14-3-3ζ protein to affect the migration and invasion ability of lung cancer cells in vivo and in vitro.In summary,these findings indicate that USP18 is a molecular pharmacologic target regulating fatty acid metabolism and metastasis.These studies set the stage of USP18 inhibitors development.