Jason J. Kwon

Pancreatic cancer: Stroma and its current and emerging targeted therapies

Pancreatic ductal adenocarcinoma (PDAC) is one of the most lethal human malignancies with a 5-year survival rate of 8%. Dense, fibrotic stroma associated with pancreatic tumors is a major obstacle for drug delivery to the tumor bed and plays a crucial role in pancreatic cancer progression. Targeting stroma is considered as a potential therapeutic strategy to improve anti-cancer drug efficacy and patient survival. Although numerous stromal depletion therapies have reached the clinic, they add little to overall survival and are often associated with toxicity. Furthermore, increasing evidence suggests the anti-tumor properties of stroma. Its complete ablation enhanced tumor progression and reduced survival. Consequently, efforts are now focused on developing stromal-targeted therapies that normalize the reactive stroma and avoid the extremes: stromal abundance vs. complete depletion. In this review, we summarized the state of current and emerging anti-stromal targeted therapies, with major emphasis on the role of miRNAs in PDAC stroma and their potential use as novel therapeutic agents to modulate PDAC tumor-stromal interactions.

Novel role of miR-29a in pancreatic cancer autophagy and its therapeutic potential

Pancreatic Ductal Adenocarcinoma (PDAC) is a highly lethal malignancy that responds poorly to current therapeutic modalities. In an effort to develop novel therapeutic strategies, we found downregulation of miR-29 in pancreatic cancer cells, and overexpression of miR-29a sensitized chemotherapeutic resistant pancreatic cancer cells to gemcitabine, reduced cancer cell viability, and increased cytotoxicity. Furthermore, miR-29a blocked autophagy flux, as evidenced by an accumulation of autophagosomes and autophagy markers, LC3B and p62, and a decrease in autophagosome-lysosome fusion. In addition, miR-29a decreased the expression of autophagy proteins, TFEB and ATG9A, which are critical for lysosomal function and autophagosome trafficking respectively. Knockdown of TFEB or ATG9A inhibited autophagy similar to miR-29a overexpression. Finally, miR-29a reduced cancer cell migration, invasion, and anchorage independent growth. Collectively, our findings indicate that miR-29a functions as a potent autophagy inhibitor, sensitizes cancer cells to gemcitabine, and decreases their invasive potential. Our data provides evidence for the use of miR-29a as a novel therapeutic agent to target PDAC.

Safety and Efficacy of AAV Retrograde Pancreatic Ductal Gene Delivery in Normal and Pancreatic Cancer Mice

Recombinant adeno-associated virus (rAAV)-mediated gene delivery shows promise to transduce the pancreas, but safety/efficacy in a neoplastic context is not well established. To identify an ideal AAV serotype, route, and vector dose and assess safety, we have investigated the use of three AAV serotypes (6, 8, and 9) expressing GFP in a self-complementary (sc) AAV vector under an EF1α promoter (scAAV.GFP) following systemic or retrograde pancreatic intraductal delivery. Systemic delivery of scAAV9.GFP transduced the pancreas with high efficiency, but gene expression did not exceed >45% with the highest dose, 5 × 1012 viral genomes (vg). Intraductal delivery of 1 × 1011 vg scAAV6.GFP transduced acini, ductal cells, and islet cells with >50%, ∼48%, and >80% efficiency, respectively, and >80% pancreatic transduction was achieved with 5 × 1011 vg. In a KrasG12D-driven pancreatic cancer mouse model, intraductal delivery of scAAV6.GFP targeted acini, epithelial, and stromal cells and exhibited persistent gene expression 5 months post-delivery. In normal mice, intraductal delivery induced a transient increase in serum amylase/lipase that resolved within a day of infusion with no sustained pancreatic inflammation or fibrosis. Similarly, in PDAC mice, intraductal delivery did not increase pancreatic intraepithelial neoplasia progression/fibrosis. Our study demonstrates that scAAV6 targets the pancreas/neoplasm efficiently and safely via retrograde pancreatic intraductal delivery.

Abstract LB-327: Restoration of MYC-repressed miR-29 in pancreatic cancer cells leads to increased reactive oxygen species and gemcitabine sensitization

Pancreatic ductal adenocarcinoma (PDAC) still remains to be one of the most highly malignant human diseases and is resistant to the majority of current therapeutic modalities. Oncogene MYC has been known to be upregulated in PDAC and plays a key role in carcinogenesis and tumor progression. In our previous work, we found the downregulation of miR-29 is a common phenomenon of PDAC, and its restored expression reduced cancer cell migration/invasive potential, anchorage independent growth, and sensitized them to gemcitabine. Furthermore, we showed that miR-29 blocks autophagy in cancer cells by targeting key autophagy related proteins. However, the mechanism associated with the loss of miR-29 in PDAC has yet to be elucidated. Here we demonstrate that MYC inhibits miR-29 expression in PDAC. MYC nuclear localization negatively correlates with miR-29 expression in various pancreatic cancer cell lines, and knockdown of MYC led to an increased expression of both primary miR-29a/b1 transcript and mature miR-29a. Furthermore, miR-29 overexpression in combination with gemcitabine led to decreased cancer cell viability and increased intracellular reactive oxygen species (ROS) and cell death. Our findings show for the first time that MYC represses miR-29 in PDAC, and miR-29 sensitizes pancreatic cancer cells to gemcitabine through increased intracellular ROS. Taken together our results, indicating the potential use of miR-29a as a novel therapeutic agent for PDAC. Citation Format: Jason J. Kwon, Kayla Quirin, Arafat Aljoufi, Amira Nafiseh, Janaiah Kota. Restoration of MYC-repressed miR-29 in pancreatic cancer cells leads to increased reactive oxygen species and gemcitabine sensitization [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2017; 2017 Apr 1-5; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2017;77(13 Suppl):Abstract nr LB-327. doi:10.1158/1538-7445.AM2017-LB-327

Abstract 172: Pathophysiological role of microRNA-29 in pancreatic cancer stroma

Proceedings: AACR 106th Annual Meeting 2015; April 18-22, 2015; Philadelphia, PA 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