Pathophysiological role of microRNA-29 in pancreatic cancer stroma

Abstract

Background: Dense fibrotic stroma associated with pancreatic ductal adenocarcinoma (PDAC) has been a major obstacle for drug delivery to the tumor bed and may impede attempts to slow down PDAC progression and metastasis. However, current antistromal drugs have not improved tumor response to chemotherapy or patient survival. Thus, a better understanding of the molecular mechanisms associated with tumorstromal interactions is desperately needed to develop novel anti-stromal therapeutic approaches. MicroRNAs (miRNAs) are an abundant class of highly conserved, small non-coding RNAs that function as key regulators of eukaryotic gene expression and cellular homeostasis. miR-29 is known to play a paramount role in the fibrotic process of several organs by providing crucial functions downstream of pro-fibrotic signaling pathways such as TGF-β1 and regulates the expression of extracellular matrix (ECM) proteins, a major component in the PDAC stroma. Upregulation of TGF-β1 is associated with PDAC pathogenesis and is known to activate stromal cells. Furthermore, vascular endothelial growth factor (VEGF) that stimulates tumor angiogenesis is a predicted target of miR-29. We hypothesize that miR-29 may be misregulated in TGF-β1 activated PDAC stromal cells and lead to excessive accumulation of ECM proteins and VEGF. Kwon et al. 2015 Annual AACR Meeting Restored expression of miR-29 could be therapeutically beneficial to modulate tumorstromal interactions. Methods: Northern blot or qPCR techniques were used to assess miR-29 levels in vitro stromal cells, murine PDAC model, and PDAC patient biopsies, and stromal deposition/fibrosis was determined by Sirius red staining. In murine and human PDAC samples, stromal specific miR-29 expression was determined via in situ hybridization by co-staining pancreatic tissues with glial fibrillary acidic protein a marker for stromal cells and miR-29. miR-29 functional studies were conducted by transfection of stroma cells with synthetic miR-29 mimics and locked nucleic acid, a miR-29 inhibitor, and ECM protein/VEGF expression was analyzed by western blot analysis. The effect of miR-29 overexpression in stromal cells on cancer colony growth was evaluated by direct coculture of stromal cells ectopically expressing miR-29 with pancreatic cancer cells, and subsequently, cancer colony number and stromal accumulation was determined by crystal violet and sirius red stains respectively. Results: In both in vitro and in vivo models as well as PDAC patient biopsies, we observed loss of miR-29 is a common phenomenon of activated stromal cells, and is associated with a significant increase in ECM and VEGF accumulation. Restored expression of miR-29 in stromal cells reduced the deposition of matrix proteins, VEGF expression, and cancer colony formation in direct co-culture. Conclusion: These results provide insight into the mechanistic role of miR-29 in PDAC stroma and its potential use as an anti-stromal/angiogenic therapeutic agent.