Comprehensive analysis of a cuproptosis-related ceRNA network implicates a potential endocrine therapy resistance mechanism in ER-positive breast cancer
Authors: Zhang Dongni , Lu Wenping , Zhuo Zhili , Wang Yanan , Zhang Weixuan , Zhang Mengfan 
Published: May 2023

Abstract
Background
While adjuvant endocrine therapy (ET) may decrease the mortality rate of estrogen receptor-positive (ER+) breast cancer (BC), the likelihood of relapse and metastasis due to ET resistance remains high. Cuproptosis is a recently discovered regulated cell death (RCD), whose role in tumors has yet to be elucidated. Thus, there is a need to study its specific regulatory mechanism in resistance to ET in BC, to identify novel therapeutic targets.

Methods
The prognostic cuproptosis-related genes (CRGs) in ER+ BC were filtered by undergoing Cox regression and least absolute shrinkage and selection operator (LASSO) regression analyses in TCGA-BRCA, and a CRGs risk signature was constructed using the correlation coefficient. Immune infiltration analysis, immune function analysis, tumor microenvironment (TME) analysis, immune checkpoint analysis, immunotherapy response analysis, drug sensitivity analysis, and pathway activation analysis were carried out among the high- and low-risk groups in turn. The central CRG of cuproptosis in ER+ BC resistance to ET was acquired through the intersection of protein interaction network (PPI) analysis, genes differentially expressed (DEGs) between human BC cells LCC9 and MCF-7 (GSE159968), and CRGs with prognostic significance in TCGA-BRCA ER+ BC. The miRNAs upstream of the core CRGs were predicted based on the intersection of 4 databases, miRDB, RNA22, miRWalk, and RNAlnter. Candidate miRNAs consisted of the intersection of predicted miRNAs and miRNAs differentially expressed in the LCC9 and MCF-7 cell lines (GSE159979). Candidate lncRNAs were the intersection of the differential lncRNAs from the LCC9 and MCF-7 cell lines and the survival-related lncRNAs obtained from a univariate Cox regression analysis. Pearson's correlation analysis was performed between mRNA-miRNA, miRNA-lncRNA, and mRNA-lncRNA expression separately.

Results
We constructed A risk signature of 4-CRGs to predict the prognosis of ER+ BC in TCGA-BRCA, a risk score = DLD*0.378 + DBT*0.201 + DLAT*0.380 + ATP7A*0.447 was used as the definition of the formula. There were significant differences between the high- and low-risk groups based on the risk score of 4-CRGs in aspects of immune infiltration, immune function, expression levels of immune checkpoint genes, and signaling pathways. DLD was determined to be the central CRG of cuproptosis in ER+ BC resistance to ET through the intersection of the PPI network analysis, DEGs between LCC9 and MCF-7 and 4-CRGs. Two miRNAs hsa-miR-370-3p and hsa-miR-432-5p were found taking DLD mRNA as a target, and the lncRNA C6orf99 has been hypothesized to be a competitive endogenous RNA that regulates DLD mRNA expression by sponging off hsa-miR-370-3p and hsa-miR-432-5p.

Conclusion
This study built a prognostic model based on genes related to cuproptosis in ER+ BC. We considered DLD to be the core gene associated with resistance to ET in ER+ BC via copper metabolism. The search for promising therapeutic targets led to the establishment of a cuproptosis-related ceRNA network C6orf99/hsa-miR-370-3p and hsa-miR-432-5p/DLD.