Abstract 3367: Overexpression of neuropilin-1 exacerbates endothelial-to-mesenchymal transition and fibrosis in pancreatic ductal adenocarcinoma

Abstract

Abstract Background: Pancreatic ductal adenocarcinoma (PDAC) is characterized by an intense fibrotic reaction termed tumour desmoplasia/fibrosis, which is partially responsible for its aggressive nature. Recently, endothelial cells have been shown to display an extreme form of plasticity that serves as an important source of fibroblasts in several pathological disorders, including cancer. The pro-fibrotic cytokine TGFβ1 promotes fibroblast accumulation via endothelial-to-mesenchymal transition (EndMT), thereby identifying EndMT as a potential therapeutic target against fibrosis. Previous studies have implicated angiogenic co-receptor, neuropilin-1 (NRP-1) also as a co-receptor for TGFβ1 in mediating several oncogenic processes. NRP-1 expression and TGFβ1 signaling have been shown to be up-regulated in PDAC. However, the driving mechanisms linking NRP-1 and EndMT in cancer remain unexplored. We therefore hypothesized that the over-expressed NRP-1 may exacerbate TGFβ1-mediated EndMT as a source of fibrosis in PDAC. Methods: NRP-1 over-expression studies were performed using lentivirus (Applied Biological Materials Inc.) in human umbilical vein endothelial cells (HUVECs), at baseline and after TGFβ1 stimulation (10 ng/mL). Total RNA was extracted using TRIzol® reagent and protein using RIPA buffer (both Invitrogen). Real-time polymerase chain reaction (RT-PCR) was performed using SYBR Green (Bio-Rad) following manufacturer's protocols. Markers of EndMT, fibrosis and TGFβ1 signaling were evaluated by RT-PCR, western blotting, Masson's trichrome staining and immunocytochemistry. Similarly, expression of NRP-1, EndMT and fibrosis markers was assessed in human PDAC xenografts to establish a correlation between NRP-1 levels with EndMT and fibrosis. Results: RT-PCR, western blotting and tube formation assay in HUVECs confirmed successful NRP-1 over-expression. TGFβ1-stimulated HUVECs demonstrated a distinct change from “cobblestone-like endothelial cell morphology” to an enlarged spindle-shaped appearance consistent with a “fibroblast-like morphology”, accompanied by rearrangement of the cytoskeleton. Moreover, over-expression of NRP-1 in HUVECs exacerbated TGFβ1-induced EndMT as demonstrated by loss of endothelial and gain of mesenchymal markers, and was further accompanied by changes in microscopic cellular characteristics consistent with EndMT. NRP-1 over-expression also up-regulated TGFβ1 signaling and expression of pro-fibrotic genes. We report herein a positive correlation between NRP-1 levels, EndMT and fibrosis markers at mRNA and protein levels in human PDAC xenografts. Conclusions: Overall, these results define a previously undetermined role of NRP-1 in regulating TGFβ1-induced EndMT and fibrosis in tumours and advocate NRP-1 as a potential therapeutic target to reduce tumour fibrosis and PDAC progression. Citation Format: Pratiek N. Matkar, Krishna K. Singh, Gerald J. Prud’homme, David W. Hedley, Howard Leong-Poi. Overexpression of neuropilin-1 exacerbates endothelial-to-mesenchymal transition and fibrosis in pancreatic ductal adenocarcinoma. [abstract]. In: Proceedings of the 107th Annual Meeting of the American Association for Cancer Research; 2016 Apr 16-20; New Orleans, LA. Philadelphia (PA): AACR; Cancer Res 2016;76(14 Suppl):Abstract nr 3367.