MYCN-Mediated Regulation Of The HES1 Promoter Enhances The Chemoresistance Of Small-Cell Lung Cancer By Modulating Apoptosis

Background:Lung carcinoma is the leading cause of cancer-related death worldwide.Small-cell lung cancer(SCLC)is an aggressive malignancy and accounts for approximately 13%-15%of all lung cancers.Although SCLC is initially sensitive to these treatments,most patients rapidly develop drug resistance,leading to chemotherapy failure.The prognosis of SCLC is very poor,with a discouraging overall survival.Therefore,it is vital to fully elucidate the molecular mechanisms of chemoresistance and discover novel effective,diagnostic and therapeutic markers for SCLCPurpose:Our previous cDNA microarray analysis showed a 2.3-fold upregulation of MYCN expression in H69AR cells compared with the expression in the parental H69 cells.Therefore,we hypothesized that MYCN may play an important role in the chemoresistance of SCLC cells.This study is to verify whether MYCN affects chemotherapy resistance of small cell lung cancer.To identify the targets of MYCN,we performed RNA sequencing(RNA-seq)and biological information analysis.Then,we further verified the relationship between downstream target genes and MYCN,and its relationship with drug resistance in small cell lung cancer.methods and results:1.MYCN is associated with multidrug resistance in small cell lung cancer in vitro and in vivo(1)CCK-8 assays showed that MYCN knockdown decreased the IC50 values of the chemotherapeutic agents(ADM,CDDP,and VP-16)in H69AR and H526 cells;(2)CCK8 assays showed that MYCN overexpression increased the IC50 values of these compounds in H69 and H446 cells.(3)subcutaneous tumor formation experiments in nude mice showed that tumors derived from H69 cells with MYCN overexpression were increased in size and showed accelerated growth in mice as well as exhibited reduced sensitization to CDDP and VP16.the H69AR cells with MYCN knockdown had smaller mean volumes and a slower rate of subcutaneous tumor growth in mice and showed significant sensitivity to CDDP and VP 16.2.MYCN affects chemoresistance in SCLC by regulating drug-induced apoptosis(1)The results of flow cytometry analysis showed that the early apoptotic fractions of MYCN-knockdown H69AR and H526 cells were significantly higher than those of siNC cells.BCL-2 expression was decreased in MYCN-downregulated H69AR and H526 cells,while BAX expression was increased.(2)The results of flow cytometry analysis showed that the early apoptotic fractions of MYCN-overexpressing H69 and H446 cells were significantly lower than those of NC cells.BCL-2 expression was increased in MYCN overexpression H69AR and H526 cells,while BAX expression was decreased.3.MYCN regulates the NOTCH pathway,and HES1 is a direct transcriptional target of MYCN(1)Through transcriptome sequencing and biological information analysis,it was found that the Notch pathway may be regulated by MYCN.Western Blot confirmed that NOTCH1,JAG2,HES1 on this pathway were positively correlated with MYCN expression.(2)CHIP-qPCR experiments and luciferase experiments confirmed that MYCN can bind to the promoter region of HES1 and promote its transcription.4.HES1 contributes to MYCN-induced chemoresistance in SCLC(1)CCK-8 assays showed that HES1 knockdown decreased the IC50 values of the chemotherapeutic agents(ADM,CDDP,and VP-16)in H69AR and H526 cells;(2)CCK-8 assays showed that the inhibition of HES1 by FLI-06 in MYCN-overexpressing cells abated the increase in the IC50 values mediated by MYCN up-regulation.(3)Subcutaneous tumor formation experiments in nude mice showed that the tumor growth and volumes could be significantly inhibited by the combination of FLI-06 and chemotherapy.5.Elevated MYCN expression correlates with poor survival and chemotherapy response in SCLC patients(1)The IHC results of the 42 SCLC patient samples showed that the samples from chemorefractory patients had a higher frequency of MYCN expression than the samples from chemosensitive patients.The MYCN levels were positively correlated with HES1 expression in the SCLC patient samples.(2)Kaplan-Meier analysis revealed that high MYCN levels were associated with poor overall survival.Conclusion:1.MYCN is positively correlated with chemoresistance in small cell lung cancer.2.MYCN regulates chemoresistance of small cell lung cancer by affecting apoptosis.3.MYCN regulates the Notch pathway and can directly bind to the HES1 promoter region to promote its transcription.4.HES1 is related to MYCN-mediated chemoresistance in small cell lung cancer.5.MYCN affects chemoresistance and survival in patients with small cell lung cancer...