Anti-tumor Effect And Mechanism Of LXR In Clear Cel Renal Cell Carcinoma By Regulating Lipid Metabolism

Background:Renal cell carcinoma(RCC)is still one of the most common malignant tumors in humans.In 2017,there were 63,900 new cases and 14400 deaths in the United States.Clear cell renal cell carcinoma(ccRCC)is the most common histological subtype of RCC,accounting for 75-80%of kidney cancer cases.Surgery is the main method of treatment,and surgical removal of localized ccRCC usually leads to better long-term disease-free survival(DFS).However,approximately 20%to 30%of patients develop metastatic ccRCC at the time of diagnosis.Besides,30%of patients with the newly diagnosed local disease have metastasis.Unfortunately,clinical outcomes such as TKI and mTOR inhibitors have not shown satisfactory improvement due to tumor recurrence and metastasis.Therefore,understanding the underlying molecular mechanisms of ccRCC and identifying new therapeutic strategies is important.Growing evidence indicates that clear cell renal cell carcinoma(ccRCC)is a disease that is related to metabolism.In particular,changes in fatty acids(FAs)and cholesterol metabolism play an important role in the development of ccRCC.As a nuclear transcription factor receptor,LXR regulates a variety of key molecules about FA synthase and cholesterol transports.So targeting LXR may provide new therapeutic targets in ccRCC.However,the potential regulatory effect and molecular mechanisms of LXR in ccRCC were still unknown.Herein,we surprisingly found that both LXR agonist and inverse agonist could inhibit proliferation,colony formation and induce apoptosis in ccRCC cells.Mechanically,we observed that LXR agonist LXR623 could downregulate the expression of cholesterol transporter LDLR while up-regulating the expression of ABCA1 which resulted in intracellular cholesterol going down and apoptosis occurring.The LXR inverse agonist SR9243 down-regulated the FA synthesis protein SREBP-1c,FASN and SCD1,causing the decrease of intracellular FA level and induce apoptosis of ccRCC cells.SR9243 and LXR623 induce apoptosis in ccRCC cells but have no killing effect on normal renal tubular epithelial HK2 cells.We also found that SRB1-mediated high-density lipoprotein cholesterol influx is the cause of high cholesterol in ccRCC cells.In conclusion,our data suggest that LXR inverse agonists and LXR agonists decreased intracellular FA and cholesterol level in ccRCC to inhibit tumor cells but have no killing effect on normal cells,LXR may be a potential safe therapeutic target for ccRCC patients.This study is divided into three parts.In the first part,we demonstrated that LXR agonist LXR623 and LXR inverse agonist SR9243 can kill tumor cells in a concentration-dependent and time-independent manner.LXR agonist LXR623 and LXR inverse agonist SR9243 can inhibit the proliferation of ccRCC cells and induce apoptosis through the endogenous apoptotic pathway.The mechanism of action of SR9243 and LXR623 was found by RNA-seq technology.SR9243 and LXR623 inhibited tumors by affecting the expression of fatty acid synthesis-related genes and the expression of cholesterol transport-related genes,thereby affecting the content of fatty acids and cholesterol in cells.Second,we verified the mRNA level and protein level of RNA-seq results,and found that the two drugs changed the cell lipid metabolism and promoted apoptosis.We also found that the two drugs have no killing effect on renal tubular epithelial cells HK2 cells,and explains the mechanism of drug safety of the two drugs.Third,we demonstrated the effect of SR9243 on the growth of 786-0 xenografts in nude mice,SR9243 inhibit tumor cells by affecting fatty acid synthesis protein,and further confirm drug safety in vivo.Objective:1.To clarify the mechanism of action of LXR agonist LXR623 and LXR inverse agonist SR9243 to inhibit cell proliferation and promote cell death.2.To clarify the molecular pathway mechanism of LXR agonist LXR623 and LXR inverse agonist SR9243 to regulate lipid metabolism in different ways.3.Determining the mechanism of LXR agonist LXR623 and LXR inverse agonist SR9243 on normal renal tubular epithelial cells.4.To clarify the anti-tumor mechanism of LXR agonist LXR623 and LXR inverse agonist SR9243 in 786-0 xenografts in nude mice.Methods:Part one1.The effect of LXR agonist LXR623 and LXR inverse agonist SR9243 on ccRCC cell proliferation was demonstrated by CCK8 cell proliferation assay.2.The effects of LXR agonist LXR623 and LXR inverse agonist SR9243 on cell proliferation and clonality were demonstrated by Edu assay and colony formation assay.3.The effects of LXR agonist LXR623 and LXR inverse agonist SR9243 on apoptosis were demonstrated by flow cytometry.The expression of endogenous apoptosis-related proteins was detected by Western Blot after LXR623 and SR9243 added.4.Through RNA-seq analysis to find the difference in mRNA expression after the action of two drugs,and analyze its potential mechanism of drug action.Part two1.Verify RNA-seq related results by qRT-PCR,such as fatty acid synthesis related genes and cholesterol transport-related gene changes2.Western Blot and cellular immunofluorescence were used to detect the changes of fatty acid synthesis related proteins and cholesterol transport-related proteins,and the intracellular triglycerides after SR9243 and LXR623 drugs were detected by triglyceride kit and cholesterol kit.3.After fatty acid was added to the cell culture dish containing SR9243,changes in fatty acid synthesis protein,cell viability,and intracellular triglyceride and lipid droplet content were observed by Western Blot,triglyceride kit,Nile red assay,and CCK8 assay.After adding cholesterol to the cell culture dish containing LXR623,the CCK8 assay was used to observe changes in cholesterol transporter,cell viability and intracellular cholesterol content by Western Blot and cholesterol kit.4.The effects of SR9243 and LXR623 on normal renal tubular epithelial cells were confirmed by CCK8 experiments.The TCGA database was used to analyze the differences in the levels of fatty acid and cholesterol synthesis and transport-related genes and endogenous LXR agonist ligand synthesis and breakdown gene mRNA in renal clear cell carcinoma and adjacent tissues.The protein expression levels of HK2,786-0 and ACHN were verified by Western Blot.The expressions of cholesterol synthesis and transporter HMGCR and SRB1 in 90 clinical and adjacent cancer specimens were verified by tissue microarray.Part three1.Construct a subcutaneous xenograft model of BALB/c nude mice.2.Divided into 2 groups:Normal saline group and SR9243 drug treatment group.On the 10th day after implanting the transplanted tumor,the nude mice were intraperitoneally injected with normal saline or SR92433.The long diameter of the subcutaneously transplanted tumor was measured weekly,the volume was calculated,and the body weight was measured.After 5 weeks,the neck was sacrificed and the tumor,blood and liver were taken.4.Immunohistochemistry to detect fatty acid synthesis protein;Oil red O staining of the tumor to detect intracellular lipid droplet content5.Biochemical liver function and renal function tests on blood samples.Result:Part one1.LXR623 and SR9243 concentration-dependent and time-dependent killing ccRCC cells2.Experiments show that LXR623 can effectively reduce the activity of cancer cells at nanomolar concentrations.Similarly,SR9243 can effectively reduce the activity of cancer cells at nanometer concentration.We performed CCK8 experiments at the same concentration for different time(12h,24h,48h,72h and 96h).We found that LXR agonist LXR623 and LXR inverse agonist SR9243 have a more pronounced effect with prolonged drug time.3,LXR623 and SR9243 can inhibit cell proliferation and clonality,Edu experiments showed that the amount and intensity of green fluorescence in the nucleus of LXR623 and SR9243 were significantly weakened.The cloning experiments showed that LXR623 and SR9243 could significantly inhibit the ability of cell clone formation.4.LXR623 and SR9243 induce apoptosis through an endogenous apoptotic pathway.Flow cytometry showed that the proportion of early apoptosis and late apoptosis was significantly increased in the LXR623 and SR9243 groups compared with the control group.Western Blot experiments showed that LXR623 and SR9243 up-regulate Bax and Cleaved-Caspase-3,down-regulate Bcl2 and induce endogenous apoptosis of ccRCC cells.5.LXR623 and SR9243 act on ccRCC cells in different ways.RNA-seq analysis showed that SR9243 can down-regulation of fatty acid synthesis related genes,while LXR623 causes cholesterol transport-related gene changes.Part two1.SR9243 down-regulation of genes related to fatty acid synthesis,and LXR623 cause changes in cholesterol transport-related genes.qRT-PCR proved that SR9243 down-regulated the fatty acid synthesis gene SREBP-lc,FASN and SCD1,while LXR623 down-regulated cholesterol influx gene LDLR and up-regulated the cholesterol efflux gene ABCA1.2.SR9243 causes a decrease in intracellular fatty acid content.LXR623 causes decreased intracellular cholesterol levels.Western Blot and immunofluorescence experiments showed that SR9243 down-regulated SREBP-1c,FASN and SCD1,and the intracellular triglyceride content were significantly decreased by triglyceride kit.LXR623 down-regulated the cholesterol influx protein LDLR,up-regulated the cholesterol efflux protein ABCA1,and the intracellular cholesterol content decreased significantly.3.SR9243 and LXR623 induce apoptosis of ccRCC cells by affecting the expression of fatty acid synthesis and cholesterol transport-related genes,thereby reducing intracellular fatty acid/cholesterol levels.After adding fatty acid to the cell culture dish containing SR9243,Western Blot,Triglyceride Kit,Nile Red Experiment,CCK8 and other experiments showed that ccRCC cells can exogenously intake fatty acids and rescue apoptosis.After adding cholesterol to the cell culture dish containing LXR623,Western Blot,cholesterol kit,CCK8 and other experiments showed that ccRCC cells can exogenously intake cholesterol and rescue apoptosis.4.SR9243 and LXR623 have no killing effect on normal renal tubular cellsCCK8 cell proliferation assay showed that SR9243 and LXR623 had no killing effect on normal renal tubular epithelial cells.TCGA database analysis of high expression of fatty acid synthesis gene in ccRCC,we found that compare with the Paracancerous tissue,ccRCC have a lower expression of cholesterol synthesis gene,lower expression of cholesterol influx gene LDLR,higher expression of cholesterol efflux gene SRB1,lower expression of endogenous LXR agonist ligand synthesis gene.tissue microarray technology showed cholesterol synthesis protein HMGCR is lowly expressed,while SRB1 is highly expressed.The protein levels of HK2,786-0 and ACHN were verified by Western Blot,and the results are consistent with the above experiments.Part three1.SR9243 inhibits the growth of transplanted tumorsThe volume and weight of transplanted tumors in the SR924 group were significantly lower than those in the control group.2.SR9243 inhibits tumors by down-regulating fatty acid synthesis proteins and reducing intracellular fatty acid content in vivo.Immunohistochemistry showed that SREBP-1c,FASN and SCD1 were significantly lower in the SR9243 group,and the oil red O experiment showed that the lipid droplet content in SR9243 group was significantly lower than that in the control group.SR9243 does not affect liver and kidney function and bodyweight of mice.The blood,liver and kidney function analysis of the mice showed that there was no significant difference between the SR9243 group and the control groupConclusion:1.SR9243 induces apoptosis in ccRCC cells by affecting the fatty acid synthesis related genes and proteins and intracellular fatty acid content.2.LXR623 induces apoptosis in ccRCC cells by affecting cholesterol-related genes and proteins and intracellular cholesterol levels.3.SR9243 induce apoptosis of ccRCC cells but have no killing effect on normal renal tubular epithelial cells,which is related to metabolic modes such as metabolic rearrangement of renal clear cell carcinoma.4.LXR623 causes apoptosis in ccRCC cells but has no killing effect on normal renal tubular epithelial cells,which is related to the lack of endogenous ligands in renal clear cell carcinoma.