The Association Between NAD+ Biosynthesis Metabolism And Immune Microenvironment In Breast Cancer Based On TCGA Omics Data

Objective: Breast cancer is the most common malignant tumor worldwide and the fifth leading cause of cancer death globally.Increasing evidence shows that tumor cells need to upregulate NAD+ biosynthesis to increase NAD+ levels to meet their growth needs.NAD+biosynthesis metabolism is related to tumor progression.However,the relationship between tumor NAD+ biosynthesis and the immune microenvironment still lacks systematic exploration.This study aimed to investigate the activity,clinical prognosis,and association with the immune microenvironment of NAD+ biosynthesis metabolism in breast cancer and pan-cancer through the data of the Cancer Genome Atlas(TCGA)and Gene Expression Omnibus(GEO),providing new direction and theoretical support for complementary therapy in cancer.Method: Part 1: Breast Cancer Molecular Subtyping Based on NAD+ Biosynthesis Genes.1.Clinical information and the gene expression profile data of breast cancer were downloaded from TCGA and GEO databases.Single-sample gene set enrichment analysis(ss GSEA)was used to quantify the NAD+ biosynthesis activity of breast cancer samples.Samples were divided into high and low NAD+ biosynthetic subtypes based on their NAD+biosynthesis activity,and survival analysis was performed.2.GSEA method was used to investigate the biological differences between the two subtypes.3.Spearman correlation analysis and ss GSEA method were used to explore the relationship between the NAD+biosynthesis score and the enrichment scores of 28 types of immune cells.4.The R package "limma" was used to analyze the differentially expressed genes(DEGs)between the high and low NAD+ biosynthetic subtypes.Immune-related genes and transcription factors were selected from DEGs to construct a transcription factor regulatory network.5.Spearman correlation analysis was used to explore the relationship between NAD+ biosynthesis activity and tumor mutational burden(TMB),homologous recombination deficiency(HRD),neoantigen,and immune checkpoints.Part 2: Construction of Breast Cancer Prognosis Model Based on NAD+ Biosynthesis-Related Genes.1.Redundant genes were removed and a prognostic risk model based on NAD+ biosynthesis-related genes was established using methods such as univariate Cox regression,lasso Cox regression,step AIC,and multivariate Cox regression analysis.2.Breast cancer samples were divided into highrisk and low-risk groups based on the risk score,and survival analysis was performed.3.The receiver operating characteristic(ROC)curve was drawn using the R package "p ROC" to evaluate the predictive ability of the model.4.The GSEA method was used to explore the biological differences between the two risk groups.5.Spearman correlation analysis was used to analyze the relationship between the risk score and TMB,HRD,neoantigen,immune checkpoint,and tumor inflammatory signatures(TIS).6.A nomogram was created based on the prognostic risk model and other independent prognostic factors obtained from multivariate Cox regression analysis.Part 3: NAD+ Biosynthesis Metabolism in Pancancer Prognosis and Immune Microenvironment.1.The gene expression profile data and survival data of 33 cancer types were obtained from the GDC Pan-Cancer(PANCAN)cohort on the UCSC Xena platform.The ss GSEA method was used to quantify the NAD+biosynthesis activity of the samples,and univariate Cox regression analysis was used to analyze the prognostic significance of NAD+ biosynthesis metabolism in pan-cancer.2.Spearman correlation analysis was used to explore the relationship between enrichment score of cancer hallmark pathways and the NAD+ biosynthesis score.3.Spearman correlation analysis and ss GSEA method were used to explore the relationship between the NAD+ biosynthesis score and enrichment score of immune signatures.4.Logistic regression was used to investigate whether NAD+ biosynthesis score can predict tumor immune signatures.5.Spearman correlation analysis was used to calculate the correlation between PD-L1 expression and NAD+ biosynthesis score in 33 cancer types.Results: 1.NAD+ biosynthesis metabolism was closely associated with the immune microenvironment and prognosis of breast cancer.Breast cancer patients with high NAD+biosynthesis score had poor prognosis,high immune infiltration,high immunogenicity and elevated PD-L1 expression,which might more benefit from immunotherapy.2.Four prognostic genes(QPRT,TFF1,CEACAM5,and MMP1)were identified by Lasso-Cox regression analysis,and a prognostic risk model was constructed.Patients in the high-risk group had poor prognosis.Univariate and multivariate Cox regression analyses showed that the risk score was an important,independent,and reliable reference factor for breast cancer prognosis.3.Pan-cancer analysis showed that NAD+ biosynthesis activity might be correlated with the active immune signatures of various cancers,and tumor with high NAD+ biosynthesis score presented an immunostimulatory tumor microenvironment.Compared with TMB,neoantigens,glycolysis,and aneuploid,NAD+ biosynthesis score was a stronger and more consistent predictor of immune signatures in different cancers.In addition,there was a positive correlation between the NAD+ biosynthesis score and PDL1 expression,cancer patients with high NAD+ biosynthesis activity might more benefit from immunotherapy.Conclusion 1.There was a significant correlation between NAD+ biosynthesis metabolism and the immune microenvironment of breast cancer.The risk score could serve as an important,independent,and reliable reference factor for breast cancer prognosis.2.NAD+biosynthesis activity might be positively associated with immune signatures in various cancers,and might serve as a biomarker for predicting the efficacy of immunotherapy.