Molecular Mechanism Of ACLY Knockdown On Ovarian Cancer Cell Proliferation And Cisplatin Resistance

BackgroundEpithelial ovarian cancer(EOC)is one of the gynecological malignant tumors with the second highest mortality rate in the world and the highest mortality rate in the United States.It is characterized by insidious onset,rapid progress,unsatisfactory tumor reductive surgery,and easy to acquire chemotherapy resistance.Most patients have progressed to advanced ovarian cancer when diagnosed.The standard treatment of patients with advanced EOC is tumor reductive surgery and platinum-based chemotherapy,but the primary and acquired resistance to platinum leads to high recurrence of ovarian cancer.Most patients with EOC respond to platinum chemotherapy,but unfortunately,tumor response to these drugs usually does not increase(or decrease)at the time of recurrence.Therefore,the development of acquired drug resistance is still one of the main factors affecting overall survival of patients with advanced EOC.ATP citrate lyase(ACLY),as a key enzyme linking glucose metabolism and lipid metabolism,converts citric acid and coenzyme A into oxaloacetic acid and acetyl coenzyme A.Acetyl CoA is the main raw material for de novo synthesis of lipoproteins and catalyzes the acetylation of proteins,especially histones.Oxaloacetic acid is the substrate for synthesis of aspartic acid,which is necessary for the synthesis of nucleotides and polyamines.It also promotes the formation of NAPDH/H+,which is involved in redox reaction and biosynthesis.Reprogramming of glycolysis and lipid synthesis are two typical changes in characteristics of cancer cells,and expression of ACLY is up-regulated in a variety of cancers,the high expression of ACLY is associated with poor prognosis in a variety of tumors.Many studies have shown that inhibition of ACLY could inhibit the proliferation,invasion,and metastasis of various tumor cells.Our previous research results showed that high expression of ACLY was associated with poor prognosis of patients with EOC,but there was no research to explore the relationship between ACLY and acquired cisplatin resistance.This study further explored the mechanism of ACLY knockdown on tumor cell proliferation,and through further bioinformatics analysis,we found that ACLY and its related pathways were significantly up-regulated in cisplatin resistant ovarian cancer cell line(A2780/CDDP).Through the construction of A2780/CDDP cell line,we verified the expression and role of ACLY in cisplatin resistant ovarian cancer cell lines,and explored the mechanism of ACLY inhibition on cisplatin resistant ovarian cancer cell lines.The specific research contents include:1.The effect and mechanism of ACLY knockdown on proliferative ability in ovarian cancer cells2.Construction and combined analysis of biological behavior changes in A2780/CDDP cell line3.The effect of ACLY inhibition on cisplatin resistance in A2780/CDDP cellsPart ? The effect and mechanism of ACLY knockdown on proliferative ability in ovarian cancer cellsObjective:1.To explore the expression and clinical significance of ACLY in ovarian cancer.2.To explore the effect of ACLY knockdown on the proliferative ability of ovarian cancer cells in vivo and in vitro and its molecular mechanism.Materials and Methods:Materials1.Bioinformatic analysis:the RNA-seqs containing comparisons between EOC and ovarian surface epithelium(OSE)tissues were searched in gene expression omnibus(GEO),and three datasets were screened(GSE18520,GSE14407,GSE36886).2.Clinical specimens:EOC tissues(N=47)were obtained from patients of Qilu hospital between September,2017 and June,2019.Patients were diagnosed as ovarian cancer by pathologic confirmation,without any chemotherapy or radio therapy.The fallopian tube epithelium(FTE)tissues of the patients with benign diseases such as uterine fibroids or uterine prolapse in the same time period were collected as control tissues.There was no statistical difference in the general conditions between the two groups of patients.3.Cell lines:The human epithelial ovarian cancer cell lines A2780,OVCAR-3,HEY,OV-90,SKOV3 and HO8910 were purchased from the Cell Bank of the Chinese Academy of Sciences(Shanghai,China).Methods1.GEO datasets containing the transcriptome sequencing of EOC and OSE tissues were downloaded and analysed by R/R studio,the different expression of ACLY was analysed between EOC and FTE tissues.The differential expression of ACLY mRNA in EOC and FTE tissues was detected by qRT-PCR.The relationship between ACLY expression and overall survival was analyzed by KM Plotter website,based on the data from TCGA and GEO databases.2.Western blot was used to detect the expression of ACLY in ovarian cancer cell lines.Stable ACLY knockdown cell lines and negative control cell lines were constructed by lentivirus transfection.The effects of ACLY knockdown on the proliferation ability of ovarian cancer cell lines(A2780,SKOV3,HEY)were detected by MTT assay and colony formation assay in vitro;the cell cycle was detected by flow cytometry;the ratio of annexin-V positive cells was detected by flow cytometry as cell apoptotic proportion;the changes of protein expression after ACLY knockdown were detected by western blot.3.In vivo experiment:A2780-NC and A2780-shACLY cells were used to construct subcutaneous tumor xenograft models to verify the change of proliferative ability induced by ACLY knockdown in vivo.Tumor sizes were measured every other day from day 10 after injection.Tumor volumes were calculated as V=[(length ×width2)/2].Results:1.The expression of ACLY in EOC tissues and its clinical significance.1.1 The expression of ACLY in EOC and OSE or FTE tissues.The RNA-seq of EOC and OSE tissue were downloaded from the GEO database.The differences in the expression of ACLY were analyzed in the datasets GSE18520,GSE14407,GSE36886.It was found that the expression of ACLY in EOC samples was higher than that in OSE samples(P<0.05,P<0.01,P<0.001 respectively)(Figure 1A-1C);qRT-PCR results showed that the expression of ACLY in EOC was higher than that in FTE tissues(P=0.0206)(Figure 1D);1.2.The clinical significance of ACLY in EOC.Bioinformatics analysis of the data showed that high expression of ACLY was associated with short overall survival,and the datasets of GSE15622,GSE18520 and GSE30161 had statistical significance(P<0.05).The statistical differences of TCGA dataset and GSE3554 dataset were P=0.051 and P=0.05 respectively(Figure 2A-2E).2.Expression of ACLY in ovarian cancer cells.Western blot results showed that ACLY expression was relatively high in A2780,SKOV3 and HEY ovarian cancer cell lines(P<0.01)(Figure 3).3.In vitro experiment to explore the effect of ACLY knockdown on the proliferative ability of EOC cells and its mechanisms.Lentivirus was used to transfect the selected three cell lines to construct ACLY stable knockdown cell line models(Figure 4).3.1 The effect of ACLY knockdown on the proliferative ability of ovarian cancer cell line.In A2780,SKOV3 and HEY cells,MTT assay showed that the proliferative ability of ACLY knockdown cells was weakened,and the difference was statistically significant(P<0.05)(Figure 5A);colony formation assay revealed that the number of clones formed in ACLY knockdown cells was significantly less than that in negative control cells,and the difference was statistically significant(P<0.001)(Figure 5B).3.2 The effect of ACLY knockdown on cell cycle changes in EOC cells.The results of flow cytometry on cell cycle showed that the proportion of cells in G0/G1 phase was up-regulated in ACLY knockdown cells(P<0.001)(Figure 6A).Western blot showed that CDK4 and CCND1 in G0/G1 phase were down-regulated(P<0.001,P<0.01)and their upstream inhibitor P16 was up-regulated(P<0.05)in ACLY knockdown cells(Figure 6B).ACLY knockdown might cause G0/G1 cell cycle arrest through P16-CDK4-CCND1 pathway.3.3 The effect of ACLY knockdown on apoptosis in EOC cells.The results of flow cytometry on apoptotic assays showed that the proportion of early and late apoptosis in ACLY knockdown cells was higher than that in the negative control cells in A2780,SKOV3,and HEY cell lines(P<0.001)(Figure 7);the results of western blot showed that cleaved PARP was up-regulated in ACLY knockdown cells,P53 was up-regulated in P53-wild-type cells like A2780 and HEY cells(P<0.05,Figure 6B)with larger scale of increase in apoptotic cells as in ACLY knockdown cells.While pan-AKT and p-AKT was down-regulated in ACLY knockdown cells.4.In vivo experiment to explore the effect of ACLY knockdown on the proliferative ability of ovarian cancer cells.The results of subcutaneous tumor xenograft models showed that the proliferative ability of A2780-shACLY cells was significantly reduced than that in A2780-NC cells,the growth curve of tumor volume of A2780-shACLY cells were significantly different(P<0.01)(Figure 8A).Xenograft tumors of A2780-shACLY cells showed reduced tumor sizes and tumor weights compared with xenograft tumors of A2780-NC cells,and the differences in tumor sizes and tumor weights were significantly different(P=0.0034)(Figure 8B,8C).Conclusions:1.ACLY was up-regulated in EOC tissues,and its high expression was associated with poor prognosis.2.ACLY knockdown inhibited tumor proliferation in EOC cells in vivo and in vitro.3.ACLY knockdown induced cell cycle arrest amd cell apoptosis in EOC cells.Part ? Construction and combined analysis of biological behavior changes in A2780/CDDP cell lineObjective:1.Through bioinformatics analysis of RNA-seq dataset in acquired cisplatin resistant ovarian cancer cell line(A2780/CDDP)and its parental cell line(A2780).2.Construction of A2780/CDDP cell line.3.Verification of the cisplatin resistance in A2780/CDDP cells and the changes of biological behavior.Materials and MethodsMaterialsThe gene expression data of A2780/CDDP cells and A2780 cells was downloaded from GEO(https://www.ncbi.nlm.nih.gov/geo/query/acc.cgi?acc=GSE15709)?Methods1.R/R Studio software was used to process the data,limma R package was used to analyze the differentially expressed genes(DEGs)of the two cell lines,the filting conditions of DEGs were:log2 | FC |?1 and P<0.05.The DEGs were enriched via clusterProfiler R package.2.A2780 cells were used to construct A2780/CDDP cells by repeated high-dose cisplatin interacting with A2780 cells;MTT assay and colony formation assay were used to explore the proliferative ability between A2780/CDDP cells and A2780 cells.MTT assay was used to detect and calculate the cisplatin resistance of the cells;western blot was used to detect the changes in molecular characteristics between A2780/CDDP cells and A2780 cells.Results1.Bioinformatics analysis of GSE15709 dataset between A2780/CDDP and A2780 cells and pathway enrichment of DEGs.According to the filtering conditions,a total number of 1648 DEGs were screened,including 640 up-regulated genes and 828 down-regulated genes in A2780/CDDP cells(Figure 1A);ACLY was up-regulated in A2780/CDDP cells(log2FC=1.42,P=0.00059)(Figure 1B);KEGG pathway enrichment showed that the DEGs were significantly enriched in PI3K/AKT pathway(P<0.05)(Figure 1C).2.Construction of A2780/CDDP cell line and exploration of the biological behavior changes.2.1 The verification of resistance to cisplatin in A2780/CDDP cells compared with A2780 cells.MTT assay and colony formation assay were used to detect the resistance to cisplatin in A2780/CDDP and A2780 cells.The IC50 of A2780/CDDP cells to cisplatin was 3 times of that of A2780 cells at 24 hours,5 times at 48 hours and 10 times at 72 hours(Figure 2A).The results of colony formation assays showed that the survival rate under same concentration of cisplatin was higher in A2780/CDDP cells,and the differences were significant(Figure 2B).2.2 Biological behavior changes of A2780/CDDP cells.The results of MTT assay and colony formation assay showed that the proliferative ability of A2780/CDDP was significantly weaker than that of A2780 cells(P<0.001,P<0.001)(Figure 3).2.3 Molecular biological changes of A2780/CDDP cells compared with A2780 cells.GSE15709 datasets showed that ACLY and PI3K/AKT pathway were upregulated and activated in A2780/CDDP cells,while AMPK was down-regulated in A2780/CDDP cells.Western blot was used to verify the changes of our new constructed A2780/CDDP cells,the changes were like those of GSE15790 dataset.The results showed that the expression of PI3K/AKT pathway molecules(PI3K,pan-AKT,p-AKT)were up-regulated in A2780/CDDP cells compared with the parental A2780 cells(P<0.001,P<0.001,P<0.01),the expression of p-AMPK-?was down-regulated(P<0.0001),which was consistent with the results of bioinformatic analysis(Figure 4A,4B).Conclusions1.Successful construction of A2780/CDDP cell line.2.Combined analysis of A2780/CDDP from GSE15709 and A2780/CDDP that we constructed showed that the expression of ACLY and PI3K/AKT pathway molecules was up-regulated in A2780/CDDP cells,while p-AMPK-? was downregulated in A2780/CDDP cells.Part ? Molecular mechanism of inhibition of ACLY regulating cisplatin resistance of A2780/CDDP cells1.To explore the effect of ACLY knockdown on cell proliferative ability in A2780/CDDP cells.2.To explore the effect of ACLY knockdown on cisplatin resistance in A2780/CDDP cells.3.To explore the mechanism of attenuating cisplatin resistance in A2780/CDDP cells induced by ACLY knockdown.Materials and Methods1.MTT assay and colony formation assay were used to detect the proliferative ability on ACLY knockdown in A2780/CDDP cells;2.MTT assay was used to detect the IC50 of cisplatin treated cells;ROS assay kit and microplate reader were used to detect the level of ROS production,ROS was detected with the 0?M and 20?M cisplatin reaction for 24 hours.Western blot was used to detect the molecular changes;In A2780/CDDP-NC cells and A2780/CDDPshACLY cells,AKT overexpression plasmid was transiently transfected with Lipofectamine 3000.Flow cytometry was used to detect cell apoptosis.3.In vivo experiment:A2780/CDDP-NC and A2780/CDDP-shACLY cells were used to construct subcutaneous tumor xenograft models to verify the change of resistance to cisplatin induced by ACLY knockdown in vivo.Tumor sizes were measured every other day from day 10 after injection.The mice received intraperitoneal injections of cisplatin at a concentration of 4 mg/kg body weight(B.W.)on days 7,14,and 28(a total of three injections).Tumor volumes were calculated as V=[(length × width2)/2].Results1.The effect of ACLY inhibition on the proliferative ability of A2780/CDDP cells.MTT assay showed that knockdown of ACLY significantly decreased the ability of cell proliferation,the difference was statistically significant(P<0.001)(Figure 1A).Colony formation assay revealed that the number of coloy in A2780/CDDP-shACLY cells were less than that of A2780/CDDP-NC cells,the difference was statistically significant(P<0.001)(Figure 1B).2.In vitro experiments to explore the knockdown of ACLY on the cisplatin resistance in A2780/CDDP cells and its mechanism.2.1 Effect of ACLY knockdown on cisplatin resistance in A2780/CDDP cells.MTT assay was used to measure the IC50 of A2780/CDDP-NC cells and A2780/CDDP-shACLY cells to cisplatin.IC50 were measured after treatment with concentration gradients of cisplatin after 24h,48h and 72h.After treatment with cisplatin for 24,48 and 72 hours,the IC50 of cisplatin in A2780/CDDP-shACLY and A2780/CDDP-NC cells were 16.64?M,31.71?M;11.55?M,26.07?M;4.648?M,14.16?M.The IC50 of cisplatin in A2780/CDDP-shACLY cells was lower than that of in A2780/CDDP-NC cells on 24h,48h and 72h(Figure 2A).The results of colony formation assay showed that under the treatment of 5 ?M and 10 ?M cisplatin,the survival ratio was calculated as compared the colony formation numbers under 5?M or 10?M cisplatin treatment with 0?M cisplatin.Comparing A2780/CDDP-shACLY cells with A2780/CDDP-NC cells,the survival rate was higher in A2780/CDDP-NC cells,which showed the less resistant to cisplatin in A2780/CDDP-shACLY cells(P<0.001)(Figure 2B).2.2 Mechanism of the effect of ACLY knockdown on cisplatin resistance in A2780/CDDP cells.2.2.1 Melacular changes on ACLY knockdown combined with cisplatin treatment.Western blot was used to measure the molecular changes between 0?M and 20?M cisplatin in A2780/CDDP-NC cells and A2780/CDDP-shACLY cells.The results revealed that PI3K,pan-AKT,and p-AKT in the PI3K/AKT pathway were downregulated following ACLY knockdown without cisplatin treatment(P<0.05),and upon cisplatin treatment,PI3K,AKT,and p-AKT were downregulated as a result of cisplatin treatment,and their downregulation was more pronounced following ACLY knockdown(P<0.05).In contrast,p-AMPK-? was upregulated following ACLY knockdown,and the increase was more obvious with cisplatin treatment and ACLY knockdown(P<0.05)(Figure 3A).2.2.2 ROS production on ACLY knockdown combined with cisplatin treatment.ROS detection assays revealed that a small increase in ROS production after knockdown of ACLY(P<0.0001).With the addition of 20?M cisplatin,the increase in ROS production was observed both in A2780/CDDP-NC and A2780/CDDPshACLY cells,but the increase in ROS production was greater in the A2780/CDDPshACLY cells(P<0.05)(Figure 3B).2.3 Effect of ACLY knockdown on apoptosis rate in response to cisplatin in A2780/CDDP cells.Flow cytometry was used to detect cell apoptosis,cells were treated with 10?M and 20?M cisplatin for 24h,48h and 72h,and the ratio of apoptotic cells in A2780/CDDP-shACLY cells was significantly higher than that in A2780/CDDP-NC cells(P<0.001)(Figure 4A).Western blot was used to detect the differences in the expression of apoptotic markers with or without treatment of cisplatin,which showed that cleaved PARP was significantly upregulated by cisplatin treatment(P<0.05),and the expression of cleaved PARP in A2780/CDDPshACLY cells was higher than that in A2780/CDDP-NC cells at the same cisplatin concentration(P<0.01),showing the same trend with the proportion of apoptotic cells in cytometry assays(Figure 4B).2.4 In vivo experiment to explore the alteration of A2780/CDDP cells resistance to cisplatin on ACLY knockdown.After intraperitoneal cisplatin injection to nude mice bearing subcutaneous tumor xenografts with A2780/CDDP-NC and A2780/CDDPshACLY cells,the volumes and weights of tumors derived from A2780/CDDPshACLY cells were significantly smaller than those of A2780/CDDP-NC cells(P<0.01)(Figure 5A,5B).3.Effects of ACLY specific inhibitor(SB-204990)combined with cisplatin in A2780/CDDP cells.SB-204990 is an ACLY specific small molecule inhibitor,and low concentrations of SB-204990(0?M,l0?M,20?M,30?M)did not affect cell proliferation,shown in MTT assay,the intergroup differences in cell proliferation ability were not statistically significant(Figure 6A).However,when cells were cotreated with cisplatin and SB-204990,the IC50 of cisplatin showed that as the concentration of SB-204990 increased,the resistance of cells to cisplatin was obvious decreased(Figure 6B).4.Effect of upregulation of AKT expression in A2780/CDDP-shACLY cells.4.1 Effect of overexpression of AKT on cisplatin resistance in A2780/CDDPshACLY cells.Afer transfection of AKT plasmid into A2780/CDDP-shACLY and A2780/CDDP-NC cells,four cells were generated:A2780/CDDP-NC-NC,A2780/CDDP-NC-AKT,A2780/CDDP-shACLY-NC and A2780/CDDP-shACLYAKT ccells.MTT assay to determine the IC50 of cisplatin on the aforementioned cells revealed that cisplatin resistance was not significantly improved in cells overexpressing AKT alone(as in A2780/CDDP-NC-AKT cells),but the decrease in cisplatin resistance upon ACLY knockdown was abolished in cells overexpressing AKT.It revealed that the decreased resistance to cisplatin upon ACLY knockdown was through downregulation of PI3K/AKT pathway(Figure 7).4.2 Effect of overexpressing AKT in ACLY knockdown cells on cell apoptosis in response to cisplatin.Flow cytometry was used to measure cell apoptosis,and the results showed that overexpression of AKT in ACLY knockdown A2780/CDDP cells resulted in a lower rate of apoptotic cells in response to cisplatin than in cell lines with ACLY knockdown alone,indicating that overexpression of AKT rescued cells from cell apoptosis partially(Figure 8A);western blot analysis of the four cell lines(A2780/CDDP-NC-NC,A2780/CDDP-NC-AKT,A2780/CDDP-shACLY-NC,A2780/CDDP-shACLY-AKT)showed that the expression of the apoptotic marker cleaved PARP was significantly upregulated by ACLY knockdown plus cisplatin,whereas it was decreased after overexpression of AKT.In cells transfected with the AKT plasmid,we detected upregulation of both AKT and p-AKT,which was statistically significantly.But as PI3K,the change of which was not significant on the overexpression of AKT(Figure 8B).Conclusions1.Inhibition of ACLY resensitizes A2780/CDDP cells to cisplatin in vitro and in vivo,possibly by inhibiting the PI3K/AKT pathway and activating the AMPK/ROS pathway.2.Specific small molecule inhibitor(SB-204990)of ACLY can alleviate cisplatin resistance in A2780/CDDP cells at low concentrations without affecting cell proliferation.