Dense fibrotic stroma associated with pancreatic ductal adenocarcinoma (PDAC) is a major obstacle for drug delivery to the tumor bed and plays a crucial role in pancreatic cancer progression. Current, anti-stromal therapies have failed to improve tumor response to chemotherapy and patient survival. Furthermore, recent studies show that stroma impedes tumor progression, and its complete ablation accelerates PDAC progression. In an effort to understand the molecular mechanisms associated with tumor-stromal interactions, using in vitro and in vivo models and PDAC patient biopsies, we show that the loss of miR-29 is a common phenomenon of activated pancreatic stellate cells (PSCs)/fibroblasts, the major stromal cells responsible for fibrotic stromal reaction. Loss of miR-29 is correlated with a significant increase in extracellular matrix (ECM) deposition, a major component in PDAC stroma. Our in vitro miR-29 gain/loss-of-function studies document the role of miR-29 in PSC-mediated ECM stromal protein accumulation. Overexpression of miR-29 in activated stellate cells reduced stromal deposition, cancer cell viability, and cancer growth in co-culture. Furthermore, the loss of miR-29 in TGF-β1 activated PSCs is SMAD3 dependent. These results provide insights into the mechanistic role of miR-29 in PDAC stroma and its potential use as a therapeutic agent to target PDAC.

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 anti-stromal drugs have not improved tumor response to chemotherapy or patient survival. Thus, a better understanding of the molecular mechanisms associated with tumor-stromal 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 regulating the expression of extracellular matrix (ECM) proteins, a major component in the PDAC stroma. Upregulation of TGF-β1 is commonly 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. Restored expression of miR-29 could be therapeutically beneficial to modulate tumor-stromal interactions. To understand the role of miR-29 in PDAC stroma, we examined miR-29 expression patterns in TGF-β1 activated stromal cells using qPCR/northern blot analysis and determined ECM and VEGF protein expression. In activated stromal cells, we observed loss of miR-29 in correlation with a significant increase in ECM and VEGF protein expression. In addition, in both murine and human PDAC samples, loss of miR-29 expression is associated with an increase in stromal percentage estimated by Sirius red stain. To evaluate the physiological role of miR-29 in stroma, we performed gain and loss-of-function studies by transfecting stromal cells with synthetic miR-29 mimics or locked nucleic acid, a miR-29 inhibitor. Overexpression of miR-29 in stromal cells suppressed matrix and VEGF protein expression, and conversely, depletion of miR-29 lead to their significant increase. Finally, to evaluate the effect of miR-29 overexpression in stromal cells on cancer colony growth, we directly co-cultured miR-29 transfected stromal cells with pancreatic cancer cells for 10 days, and subsequently, cancer colony number and stromal deposition was determined by crystal violet and Sirius red stains respectively. We observed a significant decrease in the number of cancer colonies and stromal accumulation in co-cultures. In conclusion, our results provide insight into the mechanistic role of miR-29 in PDAC stroma and its potential use as a novel anti-stromal therapeutic agent. Citation Format: Jason J. Kwon, Sarah C. Nabinger, Ravi K. Alluri, Zachary Vega, Smiti S. Sahu, Zahi Abdul Sater, Zhangsheng Yu, A Jesse Gore, Grzegorz Nalepa, Romil Saxena, Murray Korc, Janaiah Kota. Pathophysiological role of microRNA-29 in pancreatic cancer stroma. [abstract]. In: Proceedings of the 106th Annual Meeting of the American Association for Cancer Research; 2015 Apr 18-22; Philadelphia, PA. Philadelphia (PA): AACR; Cancer Res 2015;75(15 Suppl):Abstract nr 172. doi:10.1158/1538-7445.AM2015-172