The Effect On Small Cell Lung Cancer By The Circadian Protein REV-ERB Agonist SR9009 And Its Mechanisms

Background:Small cell lung cancer(SCLC)is a lung tumor with a very high degree of malignancy.Chemoresistance frequently occurs in SCLC,which leads to treatment failure.Therefore,it is necessary to explore a novel and promising therapeutic approach,which may offer hope for the discovery of new potentially useful targets for SCLC therapy.The circadian clock coordinates cell proliferation and metabolism and impacts the progression of some diseases,particularly cancer.The pharmacological modulation of the circadian machinery may be an effective therapeutic approach for treating cancer.SR9009 is a specific synthetic agonist of the REV-ERBs,essential circadian clock components.Although some studies have shown that SR9009 are specifically lethal to glioblastoma and other tumors through pharmacological modulation of REV-ERB,and it is associated with tumor autophagy,the specific molecular regulation mechanism has not been fully elucidated.However,the potential efficacy and antitumor mechanism of this drug in small-cell lung cancer(SCLC)remains poorly understood.Methods:1.We used chemosensitive cells(H69 and H446)and the corresponding chemoresistant cells(H69AR and H446DDP)to assess the efficacy of the REV-ERB agonist SR9009 for the treatment of SCLC in vitro and further validated the antitumor effect in subcutaneous tumor models of SCLC.2.Western Blot assays and Quantitative Real-time PCR assays were conducted to identify that SR9009 interacted with REV-ERB through pharmacological activation in post-translational manner.The application of small interfering RNA(siRNA)to downregulate REV-ERB? in SCLC cell lines.Cell Counting Kit-8(CCK-8)assays showed the effect of downregulation of REV-ERB?on cytotoxicity induced by SR9009.In order to explore whether REV-ERBa was involved in the antitumor effect of SR9009 in SCLC.Chromatin immunoprecipitation(Chip)sequencing assays were conducted to identify potential DNA sequences directly regulated by REV-ERBa.3.Autophagosomes were evaluated using laser confocal microscopy,transmission electron microscopy(TEM)and western blotting in our study.We sought to determine the stage of autophagy blocked by the agonist SR9009.Chloroquine(CQ)is an autophagy inhibitor that interferes autophagosomes and regulates of autophagy related genes(ATG),to determine that SR9009 induces cytotoxicity by regulating autophagy.Results:Here,we showed that the REV-ERB agonist SR9009 is specifically lethal to both chemosensitive and chemoresistant SCLC cells.REV-ERB? was involved in the antitumor effect of SR9009 in SCLC.The core autophagy gene ATG5 was identified as a direct downstream target of REV-ERBa and was suppressed by the REV-ERB agonist SR9009 in SCLC.Furthermore,the interaction of REV-ERBa with this autophagy gene impaired autophagy activity,leading to SR9009 cytotoxicity in SCLC cells.Principal conclusions:In summary,our results demonstrated for the first time that SR9009 exhibits an antitumor effect in SCLC.We identified that SR9009 interacted with REV-ERB through pharmacological activation in post-translational manner.Further research revealed that the antitumor effect of SR9009 is attributed to autophagy inhibition,which was mediated by negative regulation of the core autophagy gene Atg5 by REV-ERB?.Therefore,our research revealed that REV-ERB agonist SR9009 is a novel and promising therapeutic approach for SCLC.