Abstract 5716: miR-21 inhibition regulates mutant KRAS effector pathways and intercepts development of pancreatic ductal adenocarcinoma
Authors: Zimmerman Jacquelyn W. , Burkhart Richard A. , Chu Nina J. , Fertig Elana J. , Jaffee Elizabeth M. 
Published: September 2020

Introduction: Pancreatic ductal adenocarcinoma (PDAC) tumorigenesis is largely driven by an activating KRAS (mKRAS) mutation that is nearly ubiquitous in the earliest stages of disease, pancreatic intraepithelial neoplasias (PanINs). Targeting KRAS directly has, thus far, been unsuccessful due to compensatory pathways or associated toxicity of proposed treatments. MicroRNAs (miRNAs) are short non-coding RNAs that represent a novel avenue to target KRAS effector function through post-transcriptional regulation.

Experimental Procedures: Transgenic KrasG12D/+;Trp53R172H/+;Pdx-1-Cre (KPC) mice develop early PanINs that progress to invasive PDAC, allowing investigation into early mechanisms of tumorigenesis. The TaqMan OpenArray platform was used to globally profile miRNA expression. miRNAs of interest were validated with dedicated qPCR and inhibited with complementary antisense oligonucleotides. Effects of miR inhibition on KRAS downstream function and tumorigenicity were catalogued using cell lines, KPC, and xenograft models. The Cancer Genome Atlas (TCGA) was used to translate our findings into a human cohort. Finally, we are using patient-derived organoid models to elucidate effects on KRAS effector pathways.

Results: Global miRNA expression analysis and dedicated qPCR identified miR-21 as differentially expressed in early stage PanINs in the KPC mouse model. RNA-seq following inhibition of miR-21 revealed modulation of KRAS signaling with downregulation of MAPK, mTOR, and actin cytoskeleton pathways. Phenotypic analysis in the setting of miR-21 inhibition demonstrated reduced proliferation, migration, and invasion. Further, miR-21 inhibition delays PanIN progression to PDAC in KPC mice. Mechanistic study of miR-21 demonstrates activation of cancer associated fibroblasts (CAFs) in the tumor microenvironment (TME) and promotes resistance to chemotherapy. Analysis of TCGA PDAC cohort revealed a significant correlation between miR-21 expression and tumor epithelial cell content (P-value: hsa-miR-21-3p= 8.7e-7; hsa-miR-21-5p = 2.1e-13), prompting further studies in a human model. Organoid models with matched-isogenic CAF lines have been generated from five patients undergoing surgical resection for PDAC. Immortalized organoid models can be co-cultured with either early passage CAFs or transformed CAF lines. Organoid co-culture with CAFs are able to recapitulate the biology seen in other model systems.

Conclusions: miR-21 appears to be an early and reliable molecular marker of pancreatic neoplasia. Manipulation of miR-21 expression augments downstream KRAS effector function with phenotypic changes evident in vitro and in vivo. Organoid co-culture models are a unique and powerful tool for the study of downstream molecular mechanisms responsible for phenotypic changes resulting from inhibition of miR-21. The identification of miR-21 as regulator of KRAS effector function and tumor/CAF cross-talk reveals a potential new target for future targeted therapies.

Citation Format: Jacquelyn W. Zimmerman, Richard A. Burkhart, Nina J. Chu, Elana J. Fertig, Elizabeth M. Jaffee. miR-21 inhibition regulates mutant KRAS effector pathways and intercepts development of pancreatic ductal adenocarcinoma [abstract]. In: Proceedings of the Annual Meeting of the American Association for Cancer Research 2020; 2020 Apr 27-28 and Jun 22-24. Philadelphia (PA): AACR; Cancer Res 2020;80(16 Suppl):Abstract nr 5716.