Novel miRNA Regulation in an Early Progression Model of Pancreatic Ductal Adenocarcinoma Nina Chu1, Elizabeth M. Jaffee1 1Johns Hopkins University School of Medicine, USA Current success of immunotherapy is dependent on infiltration and function of T cells within the tumor microenvironment (TME). However, for many cancers, inflammatory and stromal cells provide formidable barriers to T cell access. Emerging data suggests that the cellular barriers to T cell access and function are regulated by both genetic and epigenetic factors. Identification of these regulators should provide new targets for enhancing immunotherapy. The goal of this research is to construct a comprehensive profile of miRNA expression within the inflammatory and stromal cells that develop in the earliest pre-malignant pancreatic intraepithelial neoplasias (PanINs) in the KrasG12D/+;Trp53R172H/+;Pdx-1-Cre (KPC) mouse model, a spontaneous model of PDA tumorigenesis. We aim to investigate the functional roles of key differentially expressed miRNAs in establishing and propagating the earliest PanIN lesions within the TME via modulating the signaling between transformed ductal epithelial cells and recruited cancer associated fibroblasts (CAFs) that comprise the majority of the desmoplastic stroma that characterizes PDA. We conducted miRNA microarray analysis to determine the levels of 750 miRNAs in the pancreata of KPC mice ranging from 4 to 12 weeks of age (pre-PanIN1 to PDA). miRNA expression was quantified by Taqman miRNA OpenArrays and confirmed by qPCR analysis. Out of the 750 miRNAs, 4 miRNAs (miR-21, miR-16, miR-19b, and miR-224) were significantly upregulated throughout PDA development. miR-21 and miR-224 are of particular interest for their regulation of targets in cancer promoting inflammatory pathways and epithelial-mesenchymal transition. Primary KPC pancreatic ductal epithelial and fibroblast cell lines were established via FACS in order to perform in vitro miRNA knock-in/out studies. qPCR of these primary cell lines show that miR-21 is significantly upregulated in KPC tumor cells and miR-224 is overexpressed in CAFs. miRNA fluorescence in situ hybridization (miR-FISH) was performed to examine the endogenous spatial expression of these two miRNAs throughout early TME progression. miR-FISH revealed that miR-21 is expressed at low levels in WT pancreata, but is highly expressed particularly in ductal epithelial cells of late stage KPC pancreata. Conversely, miR-224 is expressed by the infiltrating stromal compartment surrounding advanced lesions. Stable lentiviral inhibition of miR-21 in KPC tumor cells reduced cell proliferation, invasion, and migration, whereas overexpression of miR-224 in normal pancreatic fibroblasts increased their migratory capacity. Additional studies are underway to further determine the functional roles of these miRNAs in PDA development and progression.
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