CD81-HOTTIP Regulating Loop Enhances Chemoresistance Of Small Cell Lung Cancer Via ERK Signaling Pathway

Background and Objective:Lung cancer is still one of the most common causes of cancer-related deaths worldwide,and small cell lung cancer(SCLC)accounts for about 15%of all lung cancer cases.The high mortality of patients with SCLC is directly related to the fact that most patients eventually develop resistance to platinum-based chemotherapy and die from the disease.Therefore,there is an urgent need to determine new therapeutic targets or treatment methods to restore the sensitivity of chemotherapeutic drugs and improve the prognosis of patients with relapsed SCLC.CD81 is a member of the four-transmembrane protein family.Existing studies have found that the expression of CD81 in some tumors such as osteosarcoma and liver cancer is increased and related to the poor prognosis of patients,but the function of CD81 in SCLC is unclear.This project will explore the expression of CD81 in SCLC cells,its effect on chemotherapy response of SCLC,and related mechanisms;clarify the possibility of CD81-HOTTIP loop as a predictor of the prognosis and as a therapeutic target of SCLC.Methods:An animal model of SCLC patient-derived xenograft(PDX)and its chemotherapy resistance model were constructed,and single-cell RNA sequencing(scRNA-Seq)was used to analyze the differential genes between the chemosensitive PDXs and chemoresistant PDXs.The membrane protein,CD81,which was highly expressed in SCLC chemotherapy-resistant xenografts,was selected for subsequent research.The clinical samples of SCLC patients were collected to determine the expression of CD81 in SCLC tissues;then the value of CD81 as an assessment of chemotherapy resistance and prognosis of SCLC was evaluated.Down-regulate the expression of CD81 in chemotherapy-resistant cell lines H69AR and H446DDP,and up-regulate the expression of CD81 in chemotherapy-sensitive cell lines H69 and H446.The effect of CD81 on chemotherapy resistance of SCLC was evaluated using CCK8 assays,flow cytometry and western blotting.The effect of CD81 on chemotherapy response of SCLC xenografts was tested by subcutaneous tumor formation experiments in nude mice.qRT-PCR,western blotting,chromatin immunoprecipitation assay,and luciferase assay were used to verify the relationship between CD81,ERK signaling pathway,HOTTIP,and Myc.After interfering with the expression of CD81 and blocking the ERK signaling pathway,the effect of CD81/ERK axis on chemotherapy resistance of SCLC was evaluated.Using SCLC PDX models and nude mice subcutaneous xenograft tumor models to confirm that blocking the CD81-HOTTIP regulatory loop can increase the response of transplanted tumors to chemotherapy drugs.Results:scRNA-Seq suggest that CD81 is up-regulated in chemotherapy-resistant xenografts;CD81 is up-regulated in chemotherapy-resistant clinical specimens and chemotherapy-resistant SCLC cell lines.The high expression of CD81 is closely related to the patient’s poor prognosis.In vitro and in vivo experiments show that down-regulation of CD81 can increase the sensitivity of SCLC cells to chemotherapeutic drugs,while up-regulation of CD81 enhanced the resistance of SCLC to chemotherapeutic drugs;studies of intracellular signaling pathways have shown that down-regulation of CD81 in H69AR and H446DDP cells weakened the activity of the ERK signaling pathway,reducing the transcription of long noncoding RNA HOTTIP;rescue experiments,CHIP experiments,and luciferase experiments showed that CD81 affects the transcriptional activity of HOTTIP via ERK-β-Catenin-TCF7L2 signal axis.Strkingly,RNA pulldown experiments show that HOTTIP can promote the accumulation of Mycbp in the nucleus by binding Mycbp,and then promote the regulatory activity of transcription factor Myc on its downstream target genes including CD81.The use of small molecule inhibitors against ERK can promote the response of SCLC xenograft tumors to chemotherapy drugs.Conclusion:CD81 is highly expressed in chemotherapy-resistant cells of SCLC and is closely related to the prognosis of SCLC patients;CD81 promotes HOTTIP transcription through the ERK-β-catenin-TCF7L2 signaling pathway,which in turn can promote the transcription of CD81 through the Mycbp-Myc axis,ultimately regulate the chemothepy-resistance of SCLC.The CD81-HOTTIP regulatory loop might be used as a therapeutic target for SCLC.