SIRT1 Inhibits Chemoresistance And Cancer Sternness Of Gastric Cancer By Initiating An AMPK/FOXO3 Positive Feedback Loop

BackgroundGastric cancer is one of the most common malignant tumors in the world,and its morbidity and mortality rank among the top five in various cancers.East Asia,including China,is a high-risk area for gastric cancer,with an incidence rate of 49.2 people per 100,000 people.70%-90%of patients with gastric cancer in China have developed advanced gastric cancer at the time of treatment,and may be accompanied by peripheral invasion or metastasis.Surgical resection alone cannot be cured,so chemotherapy has become an important part of comprehensive treatment of gastric cancer.At present,most chemotherapy regimens for gastric cancer are mainly platinum,fluorouracil and other drugs,but due to the presence of gastric cancer chemoresistance,the efficacy of chemotherapy and the prognosis of patients are seriously affected.There is an urgent need to explore chemoresistance mechanisms to improve drug response.Cancer stem cells are considered to be the basis of tumor resistance and recurrence.Therefore,in-depth study of cancer stem cells can help reduce tumor stemness and enhance tumor chemotherapy sensitivity,opening up new avenues for the treatment of gastric cancer.The deacetylase SIRT1 is a type Ⅲ histone deacetylase,unlike Zn2+-dependent deacetylase,SIRT1 relies on nicotinamide-Adenine Dinucleotide(NAD).SIRT1 plays an important role in many biological processes such as metabolism,autophagy,immunity and tumors by deacetylating histone or non-histone.However,due to the type of tumor and the different signaling pathways,the role of SIRT1 in tumors is still controversial.Our previous study found that SIRT1 is lowly expressed in gastric cancer tissues.By inhibiting the transcription factor NF-κB,SIRT1 reduces the expression of the important cell cycle regulatory protein Cyclin D1,and induces gastric cancer cells to arrest in the G1 phase,thereby inhibiting the occurrence of gastric cancer.Furthermore,SIRT1 was found to bind to the transcription factor c-JUN,to deacetylate it,the transcriptional activity of c-JUN was losed,then inhibit ARHGAP5 expression,and inhibit the invasion and metastasis of gastric cancer.However,the mechanism of SIRT1 in drug resistance and sternness of gastric cancer is not cleared.The study found that hypoxia inhibits the expression of SIRT1 in non-small cell lung cancer(NSCLC),thereby inhibiting the activation of the downstream target molecule AMPK,preventing mitochondrial apoptosis,and enhancing the resistance of NSCLC cells to cisplatin and doxorubicin,and the application of SIRT1 agonists in vivo can enhance the chemosensitivity of tumor cells.Study of Sun has shown that the deacetylase SIRT1 is down-regulated in hematopoietic stem/progenitor cells of malignant proliferative myelodysplastic syndrome,thereby inhibiting the enzymatic activity of TET2 and promoting the maintenance of tumor sternness,making patients resistant to traditional therapeutic drugs.In breast cancer,the signaling pathway regulated by SIRT1 can inhibit the activity of the cell stem important transcription factor KLF4,induce the sensitivity of tumor cells to chemotherapeutic drugs,and curb tumor recurrence and metastasis.Based on this,we conducted a preliminary study on the role of SIRT1 in the drug resistance and sternness of gastric cancer cells,and found that SIRT1 is involved in the regulation of gastric cancer chemoresistance and tumor sternness.Next,we will deep research the specific function mechanism of deacetylase SIRT1 in regulating the chemoresistance and tumor sternness in gastric cancer.ObjectiveTo explore the function and regulation mechanism of SIRT1 in gastric cancer chemoresistance and tumor sternness can provide a theoretical basis and molecular targets for reducing chemotherapy resistance and recurrence of gastric cancer.Methods1.Downregulated expression of SIRT1 is related to poor prognosis of GCpatientsThe results of tissue array showed that SIRT1 was down-regulated in gastric cancer tissues.Cox regression analysis showed that the expression level of SIRT1 was significantly correlated with the survival of gastric cancer patients,survival analysis showed that patients with higher expression of SIRT1 had longer overall survival.The Kaplan-Meier Plotter database(218878_s_at)was used to analyze the survival of gastric cancer patients.It was found that gastric cancer patients with high SIRT1 expression had longer overall survival and first progression survival,and gastric cancer patients received treatment with high SIRT1 expression also had longer overall survival and first progression survival.This indicates that low expression of SIRT1 is associated with poor prognosis of gastric cancer patients,and prompts us that it is related to drug reponse.2.SIRT1 inhibits chemoresistance of GC cellsWe used stably lentivirus-infected SIRT1-overexpression and SIRT1-silencing gastric cancer cell lines(SGC-7901,BGC-823,AGS),MTS assay detected the IC50 values for cisplatin and 5-fluorouracil,and cloning forming assay detected the cloning ability of cells.We found that after inhibiting SIRT1 expression,IC50 values raised obviously,the cloning ability is enhanced.Flow cytometry and western blot were used to detect the apoptosis of cells after cisplatin treatment.The results showed that the proportion of apoptosis decreased significantly and the activity of caspase-3 decreased in SIRT1-silencing gastric cancer cell.Overexpression of SIRT1 was the opposite.This indicates that SIRT1 inhibits chemoresistance in gastric cancer cells.Nude mice were injected at a dorsal flank site with 5 × 106 cells in saline.Nude mice was divided into a cisplatin experimental group and a saline control group.Tumor volume was measured with calipers once every 7 days.When tumors reached a volume of 100 mm3,mice were injected intraperitoneally(i.p.)every 5 days with cisplatin or NaCl.On day 21,mice were sacrificed by cervicaldis location and the tumor were resected,weighed,taken pictures.It was found that cisplatin treatment can inhibited tumor volume significantly.Compared with the control group,SIRT1 overexpression tumor volume was significantly reduced,it is sensitive to cisplatin chemotherapy,but SIRT1-silencing tumor was the opposite.3.SIRT1 inhibits tumor stemness of gastric cancer cells.Using the lentivirus-infected SIRT1-overexpression and SIRT1-silencing gastric cancer cell lines,Mammosphere assay and soft agar colony formation assay were used to detect the tumor sternness,we found that overexpression of SIRT1 dramatically reduced the spheroid forming ability of GC cells,but SIRT1-silencing tumor was the opposite.Real-time PCR and flow cytometry were used to detect the expression of classical gastric cancer stem cell marker CD44 and important stem transcription factors.It was found that the mRNA levels of CD44 and stem transcription factors in SIRT1 overexpression group were decreased,and the proportion of CD44 positive cells was also significantly reduced.In suspension cultured cloning spheres and cisplatin-treated gastric cancer cells,mRNA levels of CD44 and stem transcription factors increased,while SIRT1 was down-regulated.After cisplatin treatment,CD44 positive cancer stem cells were more abundant in SIRT1-overexpression cells.4.AMPK and FOXO3 serve as the target of SIRT1 and mediate the function of SIRT1 in chemoresistance and CSC propertiesIn order to initially explore the molecular mechanism by which SIRT1 regulates gastric cancer resistance and tumor sternness,we researched on AMPK which is a downstream signal molecule of SIRT1.By measuring the IC50 of cisplatin and detecting the cell apoptosis after cisplatin treated,we found that inhibition of AMPKa in SIRT1 overexpressing gastric cancer cells can only partially reverse the chemical sensitivity induced by SIRT1.It is suggested that in addition to AMPK,there are other target genes involved in this process.We then examined whether the tumor suppressor transcription factor FOXO3 is involved in the drug sensitivity induced by SIRT1.The results showed that inhibition of FOXO3a in SIRT1 overexpressing gastric cancer cells only partially reversed the chemosensitivity induced by SIRT1,when we combined knockdown AMPKα and FOXO3a,totally reverse of improved drug response was obtained.This result suggests that both AMPK and FOXO3 serve as targets of SIRT1 in GC chemoresistance and there may be synergistic effects between these two targets.In an in vivo tumorigenicity assay,inhibitors were used to suppress AMPK and FOXO3,suppressing both AMPK and FOXO3 in SIRT1-overexpression xenograft tumors thoroughly reversed the improved cisplatin sensitivity.In addition,the regulation of AMPK and FOXO3 involved in SIRT1 on tumor stemness was confirmed by the spheroid formation.5.Positive feedback between AMPK and FOXO3Immunofluorescence staining was performed to trace the subcellular location of FOXO3a.Our results demonstrated that AMPK activator enhanced nuclear accumulation of FOXO3a in GC cells.In contrast,AMPK inhibitor demonstrated the opposite effects.Then we examined the transcriptional activity of FOXO3a using a construct containing 3 repeated FOXO-responsive elements.Data from luciferase assay showed increased transcriptional activities of FOXO3a in GC cells treated with AMPK agonist while AMPK inhibitor demonstrated the opposite effectsNext,role of FOXO3a in AMPK regulation was determined.We knocked down expression of FOXO3a in GC cells and found that mRNA levels of AMPKa decreased significantly,expression of AMPKγ did not change obviously,neither did that of AMPKβ mRNA level.We also evaluated the expression of AMPKa and AMPKy at protein levels by western blot and demonstrated that AMPKa and phosphorylation of AMPK a subunit,not AMPKγ,was downregulated after FOXO3a interference.We analyzed the promoter of AMPKα and AMPKγ with JASPAR database.Three putative binding sites of FOXO3 were found in the promoter region of AMPKa and one for AMPKγ.ChIP assay found that evident binding signals were detected in the second and third binding sites on AMPKα promoter.Only very weak band was observed for the first binding site on AMPKα promoter.And no binding signal was detected for the FOXO3 binding site on AMPKγ promoter.To further determine whether the binding of FOXO3 on AMPKα promoter has functional significance,we constructed luciferase reporter vector using promoter sequence of AMPKa containing the three FOX03 binding sites.The results revealed that FOXO3 inhibition decreased the luciferase activity driven by the AMPKa promoter.Mutation of the first binding site of FOXO3 exhibited no effects on the inhibition.Nevertheless,mutation of the second binding site of FOXO3 almost totally rescued the suppressive role of FOXO3 knockdown.Moreover,mutation of the third binding site of FOXO3 also played a role in the repressive effects.Our results indicate that both the second and the third binding sites of FOXO3 on AMPKa promoter are necessary for the binding of FOXO3 with the second site functions more important.6.Correlation among SIRT1,AMPK and FOXO3 in clinical samplesTo determine the clinical significance of AMPKα and FOX03 in GC,we assessed their expression using the tissue array including 117 pairs of paraffin-embedded GC and matched para-cancinoma tissues.Immunohistochemical staining showed that p-AMPKα and FOXO3a were underexpressed in gastric cancer tissues.The expression levels of p-AMPKα and FOXO3a in gastric cancer tissues of SIRT1 high expression group were significantly higher than those in SIRT1 low expression group.IHC scores showed that SIRT1 and FOXO3a,and p-AMPKα and FOXO3a were correlated.Survival curve results indicate that patients with high expression of p-AMPKα and FOX03a have longer survival times.Consistent with our findings,the Kaplan-Meier plotter database showed that patients with gastric cancer with high expression of p-AMPKα and FOXO3a had longer overall survival and first progression after 5-FU chemotherapy.Cox regression analysis showed that the expression levels of p-AMPKα and FOXO3a were significantly associated with survival in gastric cancer patients.ConclusionsSIRT1 can inhibit the chemoresistance and the tumor sternness of gastric cancer cells.AMPK and FOXO3 serve as targets of SIRT1 in gastric cancer chemoresistance and tumor sternness,there are synergistic effects between these two targets.