Methylation of cg09897064 and Reduced Expression of ZBP1: Innovative Checkpoint Inhibitors in Lung Adenocarcinoma with Implications for Macrophage Polarization and Tumor Growth in Lung Cancer
Lung cancer, a prevalent and aggressive disease, is characterized by recurrence and drug resistance. Understanding the underlying mechanisms and identifying new therapeutic targets are crucial for improving treatment outcomes. Our research indicates that methylation of cg09897064 and reduced expression of ZBP1 are associated with poor prognosis in lung cancer patients. Furthermore, these factors play a role in macrophage polarization, with ZBP1 upregulated in M1 macrophages compared to both M0 and M2 polarized macrophages. We observed cg09897064 methylation in M2 polarization, but not in M0 and M1 polarized macrophages. ATACseq analysis revealed closed chromatin accessibility of ZBP1 in M0 polarized macrophages, while open accessibility was observed in both M1 and M2 polarized macrophages. Our findings suggest that ZBP1 is downregulated in M0 polarized macrophages due to closed chromatin accessibility and downregulated in M2 polarized macrophages due to cg09897064 methylation. Further investigations manipulating cg09897064 methylation and ZBP1 expression through overexpression plasmids and shRNAs provided evidence for their role in modulating macrophage polarization and tumor growth. ZBP1 inhibits M2 polarization and suppresses tumor growth, while cg09897064 methylation promotes M2 polarization and macrophage-induced tumor growth. In mechanism investigations, we found that cg09897064 methylation impairs CEBPA binding to the ZBP1 promoter, leading to decreased ZBP1 expression. Targeting these factors may hold promise as a strategy for developing innovative checkpoint inhibitors in lung cancer treatment. Overall design: We performed ATAC-seq experiments with some modifications based on the protocol described by Daniel [16, 17]. M0, M1, and M2 macrophages were isolated and the number of cells was adjusted to 50 cells/ml in phosphate buffer saline (PBS). The nuclei of these macrophages were then isolated using ATAC-LB. Following DNA fragmentation and purification, the DNA was amplified through 14 cycles. To assess the fragment distribution of the libraries, an Agilent Bioanalyzer was used, and the libraries were subsequently sequenced on a HiSeq 2500 platform. We focused on ATAC-seq peaks that were located near the transcription initiation sites (TSSs) of ZBP1. These peaks were extracted and visualized in a plot showing their distribution in the reads. Additionally, motif analysis of the peak regions was conducted using the HOMER tool and the findMotifsGenome.pl function.