Publication: EPIGENETIC CHANGES MODULATED BY INHIBITOR OF KAPPA B KINASE ALPHA (IKKα) IN A PANCREATIC DUCTAL ADENOCARCINOMA (PDAC) CELL LINE
Date
2024
Journal Title
Journal ISSN
Volume Title
Publisher
International Medical University
Abstract
Background: Pancreatic ductal adenocarcinoma (PDAC) is one of the most aggressive malignancies and its recurrence, delayed diagnosis and resistance to treatments contribute to its poor survival outcomes. Epigenetic modifications such as DNA methylation significantly contribute to tumour heterogeneity, metastasis, and disease progression of PDAC. Additionally, inflammation is one of the major implications of PDAC with the nuclear factor kappa B (NF-κB) being acknowledged as its key mediator. In recent years, multiple evidence have suggested that inhibitor of kappa B kinase alpha (IKKα) plays a central role in the initiation and progression of PDAC through transcriptional activity that enhances the NF-κB-mediated gene expressions. However, there has been lack of evidence correlating the role of IKKα and its epigenetic changes in PDAC.
Aim: To determine the epigenetic changes associated with IKKα in a PDAC cell line. To identify gene pathways that are regulated by IKKα in the PDAC cell line.
Methodology: Methylome-wide analysis was conducted on two cell lines – the parental PANC1 cell line PANC1_WT and PANC1_33 with the IKKα knockout. Briefly, DNA extracted from both cell lines were subjected to bisulfite conversion and DNA methylation using the Illumina Infinium MethylationEPIC BeadChip array was performed to identify the differentially methylated sites between both cell lines. Pathway enrichment analysis was conducted using the identified differentially methylated regions using EnrichR to gather information on the signaling pathways and genes with altered patterns of DNA methylation in the IKKα knockout cells relative to the parental cell lines. The top 5 most significant hypomethylated and hypermethylated regions were enriched for genes involved in ERBB2 regulation. qRT-PCR was performed to confirm our methylation data and determine the ERBB2 expression in the PDAC cell line. Other confirmatory tests such as in vitro cell assays comprising of cell proliferation and cell migration were also performed to further study the characteristics of the cell lines. Statistical analysis using the EnrichR and Student’s t-test value were performed using the mean and standard deviation values. Findings were deemed statistically significant if p<0.05.
Results: Genomic regions that were hypermethylated in the IKKα knockout cells relative to the parental cell lines were significantly enriched for genes involved in ERBB2-related signaling pathways such as the PTK6-ERK/MAPK, and PI3K events. The qRT-PCR confirmed the downregulation of ERBB2 expression in the IKKα knockout cell line. A decrease in the cell proliferation rate was also observed when compared to the wildtype. There were no statistically significant results obtained from the hypomethylated regions and cell migration assay.
Conclusion: Perturbations to IKKα expression result in epigenetic alterations in PDAC cell line via the NF-κB dependent and/or independent pathways. Subset genes such as NRG1, NRG3, ERBB4, and EGFR were also identified to be regulated by IKKα.
Key words: IKKα, NF-κB, epigenetics, DNA methylation, PDAC
Description
Keywords
Epigenomics, DNA Methylation, Adenocarcinoma, Cell Line, Pancreatic Neoplasms