ACSS3 Represses Prostate Cancer Progression Through Downregulating Lipid Droplet-associated Protein PLIN3

Prostate cancer(PCa)is an androgen-dependent tumor,and androgen can stimulate PCa cell growth and disease progression.Traditional endocrine therapies include androgen deprivation therapy(ADT)that blocks testicular androgens and antiandrogen therapy that block adrenal androgens.Traditional endocrine therapies include ADT that blocks testicular androgens and antiandrogen therapy that block adrenal androgens.However,due to increased intratumoural androgen synthesis and AR variation,PCa progresses to castration-resistant prostate cancer(CRPC),which ultimately becomes resistant to endocrine therapy.A search for new therapeutic perspectives is urgently needed.Abnormal lipid accumulation has been found in many tumor cells,such as clear cell renal carcinoma,breast cancer and PCa.Some studies have shown that lipid accumulation in PCa cells is closely related to the development of CRPC and Enzalutamide or Abiraterone resistance.Effective ways to reduce lipid accumulation in PCa remain unclear.In this study,by screening lipid metabolism-related gene sets and bioinformatics analysis in prostate cancer database,we identified the key lipid metabolism-related gene ACSS3 in PCa.We found that ACSS3/PLIN3 signaling inhibits PCa progression through increasing ER stress and reverses Enzalutamide resistance by reducing intratumoral LD deposits.Mechanistic investigations demonstrated that ACSS3 reduced LD deposits by regulating the stability of the LD coat protein PLIN3.Together,these results establish ACSS3 inhibits prostate cancer progression and new endocrine therapy resistance by reducing intratumoral lipid accumulation.Our study includes the following five parts:Part ?: Screening and expression of lipid metabolism related genes in prostate cancer Objective: To identify the key genes related to lipid metabolism in PCa gene expression data.And to explore the relationship between target gene expression and clinical information in PCa.Methods: Collecting the bioinformation data of prostate cancer from TCGA and GEO databases,and screening the key lipid metabolism-related genes with significant statistical differences between cancer and normal tissues,according to lipid metabolism-related gene sets from Oncomine database.Then the correlation between key gene and pathological stage,Gleason score,prognosis and other clinical information of prostate cancer were further clarified.Immunohistochemistry(IHC)assay was used to detect the expression of key gene in clinical samples of PCa.The publicly available Cancer Cell Line Encyclopedia(CCLE)database was used for bioinformatic analyses to evaluate the methylation status of the ACSS3 promoter in different PCa cell lines.Compare the methylation status of the ACSS3 promoter in PCa cells and in normal prostate cells by bisulfite sequencing PCR(BSP).To determine whether DNA methylation is responsible for ACSS3 downregulation,we treated 22RV1,PC3 and C4-2 cells with or without 5-aza-d C,a demethylation reagent.Results: By screening lipid metabolism ?related gene sets in five independent PCa databases(TCGA Prostate,Taylor Prostate,Lapointe Prostate,Tomlins Prostate,Grasso Prostate),four genes were selected;three of these genes(ACSS3,ACSF2 and CLU)were found to be downregulated and one gene(FABP5)was found to be upregulated in PCa compared to normal prostate tissues(|Log FC| > 1.5,p < 0.05).The patients with tumors that only expressed high levels of ACSS3 had longer DFS(p = 0.0007)and BRFS(p =0.0072)than patients with tumors expressing low levels of ACSS3.Low ACSS3 expression was positively correlated with tumor stage,metastasis,recurrence and Gleason score.The result of IHC demonstrated that ACSS3 protein levels were significantly lower in the PCa tissues(n = 371)than in the normal tissues(n = 107),and low ACSS3 expression was positively correlated with tumor stage and Gleason score.Methylation was enriched in the ACSS3 promoter.Hypermethylation of the ACSS3 promoter was found in PCa cells compared with normal prostate cells,and hypermethylation plays a crucial role in the silencing of ACSS3 expression.Conclusion: ACSS3 was downregulated and predicted poor prognosis in PCa.Loss of ACSS3 expression was due to gene promoter methylation.Part ?: The role and mechanism of ACSS3 in PCa Objective: To explore the biological and mechanism of ACSS3 in PCa.Methods: We successfully generated C4-2 and 22RV1 cell lines with overexpression or knockout of ACSS3 by using lentivirus or sg RNA(CRISPR-Cas9)analysis.CCK-8,invasion assays,Oil Red assay,liquid chromatography/mass spectrometry(LC/MS)lipidomic assay,RNA sequencing were conducted to explore the biological and mechanism of ACSS3 in PCa.Results: The cells stably overexpressing ACSS3 had significantly reduced proliferation,migration and invasion,while ACSS3-knockout cell lines tended to display increased proliferation,migration and invasion.And the important components of LDs were significantly downregulated in stable ACSS3-overexpressing C4-2 cells compared to control cells.Further research found that ACSS3 promoted ER stress-mediated cell apoptosis.Conclusion: ACSS3 promoted ER stress-mediated cell apoptosis,inhibited abnormal lipid accumulation and tumor cell growth.Part ?: ACSS3 regulated the protein stability of PLIN3Objective: To investigate the mechanism of ACSS3 in reducing lipid accumulation in PCa cellsMethods: We used q RT-PCR and Western blot to examine the m RNA and protein levels of LD-associated protein in 22RV1 and C4-2 cells with overexpression or knockout of ACSS3.Accordingly,22RV1 cells stably overexpressing ACSS3 or with stable knockout of ACSS3 were treated with cycloheximide(CHX)to inhibit protein synthesis,and PLIN3 protein turnover was analyzed over time.To further explore which pathway played a major role in the process,a proteasome inhibitor(MG132)and a lysosome inhibitor(chloroquine)were administered to ACSS3-overexpressing cells.Ubiquitination?related immunoprecipitation was used to assess the levels of ubiquitinated PLIN3.Then we stably overexpressed ACSS3 with overexpression of PLIN3 in PCa cells.CCK-8 assay,migration assay,oil red staining,and Western blotting were performed.Meanwhile,Immunofluorescent was performed to detect the influence of ACSS3 in PLIN3 levels and LDs.Results: The rt-PCR results showed no statistically significant changes in the m RNA levels of PLIN2 and PLIN3.But overexpression of ACSS3 in C4-2 and 22RV1 cells resulted in decreased PLIN3 protein levels,and knockout of ACSS3 in 22RV1 cells resulted in increased PLIN3 protein levels compared to those in control cells.Further research found that ACSS3 recruits AIP4 E3 ubiquitin ligase to lipid droplets and by this means regulates the level of ubiquitination of PLIN3.Compared with the Con+NC,overexpressed PLIN3 alone or ACSS3+PLIN3 promoted cell proliferation and migration,increased LD deposit size and TG and cholesterol contents.Then compared with overexpressed PLIN3,overexpressed ACSS3+PLIN3 did not affect cell proliferation,migration,LD deposition area,TG and cholesterol content.Conclusion: ACSS3 regulated the protein stability of PLIN3,which rescued the ACSS3-mediated tumor cell suppression function.Part ?: ACSS3 inhibited CRPC progression and reversed Enzalutamide resistanceObjective: To investigate the effect and mechanism of ACSS3 on the progression of prostate cancer and enzalutamine resistanceMethods: LNCa P,22RV1 and C4-2 cells stably overexpressing ACSS3 were treated with cholesterol.We conducted LC/MS to evaluate androgen synthesis in tumor cells,which was the level of testosterone(T)and dihydrotestosterone(DHT)contents in C4-2 cells stably overexpressing ACSS3 and/or knocking down PLIN3.C4-2-ENZR was constructed to detect the changes of ACSS3 expression level and intracellular lipid content before and after drug resistance.CCK8 assay was used to detect the sensitivity of enzalutamide-resistant cell lines 22RV1 and C4-2-ENZR with overexpressing ACSS3.The effect of ACSS3 on enzalutamide resistance in prostate cancer was studied in vivo by constructing stable overexpressing ACSS3 enzalutamide resistant cell lines.Results: Overexpression of ACSS3 inhibited LNCap and 22RV1 cell proliferation,while cholesterol treatment alleviated the ACSS3-mediated suppression,and the overexpression of ACSS3 restored the sensitivity of C4-2 cells to cholesterol treatment.Restoration of ACSS3 expression significantly decreased the T and DHT contents in C4-2 cells and the effects of ACSS3 expression was mediated through downregulation of PLIN3.Overexpression of ACSS3 restored the sensitivity of 22RV1 and C4-2-ENZR cells to enzalutamide treatment.Conclusion: ACSS3 repressed CRPC progression,reversed Enzalutamide resistance...