Abstract
Due to abundant stroma and extracellular matrix, accompanied by lack of vascularization, pancreatic ductal adenocarcinoma (PDAC) is characterized by severe hypoxia. Epigenetic regulation is likely one of the mechanisms driving hypoxia-induced epithelial-to-mesenchymal transition (EMT), responsible for PDAC aggressiveness and dismal prognosis. To verify the role of DNA methylation in this process, we assessed gene expression and DNA methylation changes in four PDAC cell lines. BxPC-3, MIA PaCa-2, PANC-1, and SU.86.86 cells were exposed to conditioned media containing cytokines and inflammatory molecules in normoxic and hypoxic (1% O2) conditions for 2 and 6 days. Cancer Inflammation and Immunity Crosstalk and Human Epithelial to Mesenchymal Transition RT² Profiler PCR Arrays were used to identify top deregulated inflammatory and EMT-related genes. Their mRNA expression and DNA methylation were quantified by qRT-PCR and pyrosequencing. BxPC-3 and SU.86.86 cell lines were the most sensitive to hypoxia and inflammation. Although the methylation of gene promoters correlated with gene expression negatively, it was not significantly influenced by experimental conditions. However, DNA methyltransferase inhibitor decitabine efficiently decreased DNA methylation up to 53% and reactivated all silenced genes. These results confirm the role of DNA methylation in EMT-related gene regulation and uncover possible new targets involved in PDAC progression.
Highlights
Pancreatic ductal adenocarcinoma (PDAC), representing more than 90% of all pancreatic cancers, is estimated to become the second leading cause of cancer-related deaths in developed countries by 2030 [1]
Four pancreatic ductal adenocarcinoma (PDAC) cell lines, BxPC-3, MIA PaCa-2, and PANC-1, derived from the primary adenocarcinoma, and SU86.86 cells derived from liver metastasis, were used to assess EMTrelated changes in gene expression and DNA methylation
Unique molecular features, including epithelial-mesenchymal phenotype and neuroendocrine differentiation attributed to MIA PaCa-2 and PANC-1 [34], together with divergent genetic profiles, may be responsible for a distinct response of the studied cell lines to experimental conditions [35]
Summary
Pancreatic ductal adenocarcinoma (PDAC), representing more than 90% of all pancreatic cancers, is estimated to become the second leading cause of cancer-related deaths in developed countries by 2030 [1]. Only 11% of PDACs are detected early, with a 5-year survival rate of. If cancer has spread to surrounding tissues or organs, a 5-year survival rate drops down to 13%, and for the 52% of patients diagnosed in the late stage, it decreases to only 3% [2]. Accumulation of genetic, epigenetic, and morphological changes in pancreatic ductal cells is causal in disease initiation and development. The progression from hyperplasia through dysplasia to invasive PDAC is at the molecular level associated with telomere shortening and accumulation of mutations in KRAS, ERBB2, CDKN2A in low-grade, and TP53, SMAD4, and BRCA2 in high-grade pre-invasive precursor lesions [3]. While genetics is critical for PDAC initiation and early progression, the acquisition of tumor heterogeneity is associated with specific epigenomic landscapes [5,6]
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