Multi-omics Characterization Of Autophagy-related Molecules In Cancer Therapy

Research Background: Autophagy is a highly conserved lysosomal degradation and metabolic process that enables cells to maintain homeostasis by recycling nutrients from damaged organelles and proteins.Autophagy plays a complex role in cancer at the molecular level,including genomics,transcriptomics,and proteomics.Autophagy has a dual effect in the treatment of cancer,depending on various factors such as tumor type,staging,tumor microenvironment,and genetic background,therefore targeting autophagy may be a new strategy for treating cancer.Melanoma is a malignant tumor originating from melanocytes,with the most common site of occurrence being the skin,accounting for over 90%of melanoma cases.Melanoma accounts for only about 1% of all skin tumors,but it is the most severe and deadly type of skin tumor.Treatment options for melanoma include surgical resection,chemotherapy,immunotherapy,targeted therapy,biochemical therapy,and photodynamic therapy,which can be used individually or in combination based on the patient’s health status,disease staging,genetic characteristics,and tumor location.However,the response rate to immunotherapy in advanced melanoma patients is not high,and targeted drugs are prone to drug resistance,indicating an urgent need to develop new treatment methods to improve the treatment outcomes for melanoma patients.Research Objectives:(1)To establish a tumor autophagy status evaluation system,and to analyze the effect of autophagy on tumor molecular characteristics and drug sensitivity using multi-omics data analysis.(2)To test the sensitivity of melanoma to anticancer drugs after inducing autophagy in vitro and in vivo experiments.(3)To explore the molecular mechanisms underlying the enhancement of melanoma’s sensitivity to anticancer drugs induced by autophagy.Research Methods:(1)Single-sample gene set enrichment analysis method to screen autophagy-related gene sets from autophagy-related databases and independent datasets.(2)Cell proliferation experiments to test melanoma cell viability.(3)Flow cytometry to detect melanoma cell death.(4)Western blot to detect protein expression levels.(5)q RT-PCR to detect m RNA expression levels of target genes.(6)RNA-seq to detect changes in cell transcriptomics.(7)Establishing a subcutaneous tumor model in nude mice to study the effects of different interventions on mouse melanoma growth.(8)Constructing gene knockdown and overexpression cell lines through plasmid transient transfection and lentivirus packaging experiments.Research Results:(1)Through data analysis of autophagy-related databases and independent datasets,a 37-gene autophagy feature gene set was screened and a tumor autophagy status evaluation system was successfully established.(2)By integrating large-scale tumor multi-omics data and public drug response data from the cancer genome atlas(TCGA),the autophagy status of approximately 10,000 tumor samples from 33 tumor types in TCGA was evaluated using the established autophagy evaluation system.The multi-omics changes(m RNA,mi RNA,proteins,somatic mutations,and somatic copy number alterations)in different tumor types under different autophagy statuses were compared,and the autophagy-related multi-omics molecular patterns in multiple tumor types were described.(3)Based on the autophagy score and drug response data of tumor samples from TCGA for 138 drugs,the correlation between tumor autophagy status and drug sensitivity was analyzed.In contrast to the traditional view that inducing autophagy leads to drug resistance in cancer treatment,our results found that inducing autophagy can make tumors more sensitive to drugs,indicating the complex effects of autophagy on drug response.(4)Through cell experiments,it was found that in the A375,SK-MEL-28,and SK-MEL-5 melanoma cell lines,under two autophagy induction conditions of Rapamycin and EBSS,the relative cell viability of melanoma cells decreased and cell death increased after treatment with Etoposide and BMS536924.In vitro experiments showed that melanoma cell lines became significantily sensitive to Etoposide and BMS536924 under autophagy inducer.(5)Through a subcutaneous tumor experiment in nude mice,it was found that compared with the Vehicle group,the growth rate of melanoma was slower in the Rapamycin group and the Etoposide group.The growth rate of melanoma was slowest in the Rapamycin+Etoposide combination therapy group.In vivo experiments showed that inducing autophagy enhanced melanoma sensitivity to Etoposide.(6)RNA-seq analysis and q RT-PCR results showed that compared with the control group,the expression level of DDIT4 decreased in the Rapamycin group and the Etoposide group,and the expression level of DDIT4 was the lowest in the Rapamycin+Etoposide combination therapy group,suggesting that DDIT4 may be a key mediator molecule.(7)By knocking down and overexpressing DDIT4 in melanoma cells,it was proved that melanoma cells may be more sensitive to Etoposide when present with an autophagy inducer,and this pattern may be mediated through DDIT4.Research Conclusions:(1)The autophagy feature gene set consisting of 37 genes was determined,and a tumor autophagy status evaluation system was established.(2)Autophagy-related multi-omics molecular patterns in multiple tumor types were described,providing strong biological support for autophagy research.(3)The correlation between tumor autophagy status and drug sensitivity was analyzed,and it was found that inducing autophagy can make tumors more sensitive to anticancer drugs,and provides more options for tumor therapy targeting autophagy.(4)Inducing autophagy can sensitize melanoma cell to anticancer drugs,such as Etoposide and BMS536924.(5)Rapamycin-induced autophagy enhances melanoma sensitivity to Etoposide,and DDIT4 is one of the key mediator molecules in this process.It provides a new target for tumor therapy.