| BackgroundMetabolic syndrome(MS)is mainly characterized by obesity,glucose metabolic disorder,hypertension and dyslipidemia.The incidence of MS has been increasing with socioeconomic development and change of everyday diet.Metabolic abnormality not only has close association with cardiovascular diseases,but also contributes to the oncogenesis and progression of multiple types of cancer including breast,liver,pancreatic,colorectal,endometrial and bladder cancer.Although metabolic diseases exert influence on human health,researches on tumor metabolism are crucial for the development of anti-metabolic drugs and strategies for cancer treatment.Hepatocellular carcinoma(HCC)is the sixth most common malignancy worldwide and the third leading cause of cancer-related mortality.Common risk factors of HCC consist of viral hepatitis,alcoholic liver disease,non-alcoholic fatty liver disease and aflatoxin.In recent years,non-alcoholic fatty liver disease has become the primary cause of HCC in developed regions due to the effective prevention and control of viral hepatitis.It has become a hot topic to explore new strategies of HCC treatment from the perspective of cancer metabolism.Currently,some anti-metabolic drugs have been proven to efficaciously inhibit liver cancer growth and metastasis by regulating cell cycle,signal transduction and liver metabolism.Therefore,further study on the underlying anti-tumor mechanism of anti-metabolic drugs may lead to major breakthroughs in the prevention and treatment of HCC.Cholesterol plays a significant role in the formation and function of cell membrane.Hypercholesterolemia has been confirmed as a risk factor for cardiovascular and cerebrovascular diseases,while the relationship between total serum cholesterol(TSC)level and cancer occurrence and prognosis is still controversial.Notably,several epidemiological studies and meta-analyses have indicated that high cholesterol levels can reduce the risk of HCC occurrence and improve the outcomes of HCC patients.However,most studies attributed this phenomenon to impaired liver function in patients with lower cholesterol levels and the detailed mechanism of the role of cholesterol in HCC is unclear.In this study,we explored the impact and mechanism of cholesterol on the prognosis of HCC patients.Methods1.Clinicopathologic data of 969 patients underwent hepatectomy in our hospital were retrospectively analyzed.The included patients were divided into the high and low serum cholesterol level groups based on the calculated cut-off value.The relationship between serum cholesterol and prognosis was explored using log-rank test and Cox proportional hazards regression model.Mann-Whitney non-parametric test or chi-square test was used to analyze the correlation between cholesterol and clinicopathologic characteristics.2.The effect of TSC level on HCC invasion and metastasis in vivo was investigated.Six-week-old severe combined immunodeficient NCG mice were fed a high-cholesterol diet(HCD)for 4 weeks to mimic high serum cholesterol levels in patients.Liver and lung metastasis model were respectively established through injecting SMMC-7721 cells into the spleen and HCC-LM3 cells into the tail vein of mouse.Meanwhile,CCK8 and real-time apoptosis detection kit were used to detect cell proliferation and apoptosis after cholesterol treatment.The effect of cholesterol on the invasion and metastasis of HCC was then tested by transwell and matrigel invasion chambers in vitro.3.To confirm whether the effect of cholesterol on tumor invasion and metastasis was achieved through epithelial-mesenchymal transition(EMT),typical EMT-related molecular markers,such as w-catenin,E-cadherin,Vimentin and Slug,were detected by Western blot.Cholesterol quantitative kit and immunofluorescence analysis were further applied to measure the changes of cellular cholesterol and the amount of lipid rafts on cell membrane before and after treated with cholesterol.Readyprep protein extraction kit was used to separate lipid and non-lipid raft fractions,and the expression and location of important molecules involved in invasion and metastasis were examined before and after cholesterol treatment.4.Western blot was applied to reveal the effect of cholesterol treatment on CD44 content in lipid and non-lipid raft fractions of HCC cell lines.The co-localization of CD44 and lipid rafts was observed by immunofluorescence assay.The lipid and non-lipid raft components were isolated from samples of HCC patients with high and low serum cholesterol level,and the expression and location of CD44 in the two components were detected by western blot.5.The binding of CD44 and Ezrin was evaluated in cell lines,samples of mice models and HCC patients by immunoprecipitation.The co-localization of CD44 and Ezrin was then further observed by immunofluorescence assay to determine the effect of cholesterol on binding of CD44 to Ezrin.6.The impact of CD44-Ezrin pathway on HCC invasion and metastasis was further investigated by intervening the expression of CD44,inhibiting and promoting CD44 localization into lipid raft.siRNA was used to down-regulate the expression of CD44 in HCC cell lines.The palmitoylation inhibitors(PI)was applied to prevent CD44 from entering lipid rafts,while the depalmitoylation inhibitors(DPI)were used to simulate the effect of cholesterol treatment.Results1.Serum cholesterol level was associated with the prognosis of HCC patients,and high TSC level served as a protective factor for overall survival(OS)and disease-free survival(DFS).On the premise of similar liver function,tumor diameter,number and differentiation,TSC was correlated with microvascular invasion and lymph node metastasis.2.Both liver and lung metastasis models showed that HCD treatment resulted in a remarkable reduction of the number and the maximum size of metastatic lesions.Meanwhile,although proliferation and apoptosis of HCC cell lines were not regulated by cholesterol,the invasion and migration ability were restrained after cholesterol treatment in vitro.3.Although exerting no influence on EMT and typical cellular signaling pathways,cholesterol altered the localization of relevant molecules on cell surface.Cholesterol administration accelerated lipid raft formation and facilitated CD44 translocation into lipid rafts.Consistently,more localization of CD44 was detected in lipid rafts of HCC samples from patients with high TSC levels.4.Cholesterol-induced CD44 translocation to lipid rafts led to a dramatic decrease in CD44-Ezrin interaction.HCC samples showed lower binding capacity of CD44 to Ezrin in patients with high TSC compared with that in patients with low TSC.Moreover,decreased co-localization of CD44 with Ezrin on the tumor cell membrane was observed in metastatic foci from the liver and lung in HCD mice.5.Down-regulation of CD44 decreased the expression in both lipid and non-lipid raft.CD44 knockdown dramatically reduced cell migration and invasion.PI decreased CD44 localization into lipid rafts of HCC cells,promoted the binding of CD44 with Ezrin,and consequently blocked the inhibitory effect of cholesterol on cell migration and invasion.Similar to cholesterol treatment,DPI sequestered more CD44 in lipid rafts and inhibited the interaction between CD44 and Ezrin,and then remarkably suppressed HCC cell migration and invasion.ConclusionIn conclusion,our study demonstrated that high serum cholesterol level predicts better outcomes in HCC patients.High cholesterol combats HCC metastasis by favoring lipid raft formation and CD44 translocation into rafts.Targeting CD44-Ezrin signaling is therefore a potential approach to improve HCC prognosis. |
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