Background: Pancreatic ductal adenocarcinoma (PDAC) is a deadly disease driven by mutated KRAS with an average survival of only 3-6 months from the time of diagnosis. Interferon-Stimulated Gene 15 (ISG15) is a 15 kDa protein induced by Type I interferons (IFN-α and IFN-β) and is a member of the ubiquitin-like superfamily of proteins. The ISG15 pathway is highly elevated in various malignancies where it has been shown to stabilize KRAS protein expression. However, very little is known about the role of the ISG15 pathway in PDAC. The purpose of this project is to investigate the expression and proliferative effects of the ISG15 pathway in PDAC.
Methods: The ISG15 knockdown cells were created using the CRISPR (... More
Background: Pancreatic ductal adenocarcinoma (PDAC) is a deadly disease driven by mutated KRAS with an average survival of only 3-6 months from the time of diagnosis. Interferon-Stimulated Gene 15 (ISG15) is a 15 kDa protein induced by Type I interferons (IFN-α and IFN-β) and is a member of the ubiquitin-like superfamily of proteins. The ISG15 pathway is highly elevated in various malignancies where it has been shown to stabilize KRAS protein expression. However, very little is known about the role of the ISG15 pathway in PDAC. The purpose of this project is to investigate the expression and proliferative effects of the ISG15 pathway in PDAC.
Methods: The ISG15 knockdown cells were created using the CRISPR (Clustered Regularly Interspaced Short Palindromic Repeats) gene editing system. ISG15 was stably knocked-down in murine Panc02 pancreatic cancer cells by transfection with an ISG15 CRISPR construct from Genscript (pSpCas9 BB-2A-Puro (PX459)) and treated with media containing puromycin (2 μg/ml) for selection. Knockdown was confirmed by RT-PCR. The effect of ISG15 knockdown on PDAC cell growth in vitro was determined by comparing viable cell counts on wild-type and ISG15 knockdown Panc02 cells 48 hours after plating. The effect of ISG15 knockdown on pancreatic tumorigenesis was determined by subcutaneous inoculation of syngeneic immune competent C57BL/6 mice with 1x106 Panc02 wild-type or ISG15 knockdown cells. Tumor volumes were measured weekly and after 3 weeks the mice were euthanized, tumors dissected and weighed.
Results: ISG15 CRISPR treated cells had a 50% reduction in ISG15 mRNA expression as compared to wild-type cells. ISG15 knockdown in Panc02 PDAC cells significantly reduced cell growth as compared to wild-type cells (p=0.030). Tumors formed from ISG15 knockdown Panc02 cells significantly had a 74% reduction in tumor volume (p=0.031) and 78% reduction in tumor weight (p=0.029) as compared to wild-type cells.
Conclusion: The ISG15 pathway contributes to PDAC by increasing cell growth and the tumorigenic potential of pancreatic cancer cells. Ultimately, strategies to target the ISG15 pathway may lead to improved survival for patients diagnosed with PDAC.
