ISG15 pathway knockdown reverses pancreatic cancer cell transformation and decreases murine pancreatic tumor growth via downregulation of PDL-1 expression
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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 expressed in various malignancies, including pancreatic ductal adenocarcinoma (PDAC), suggesting a potential role of the ISG15 pathway (free ISG15 and ISG15 conjugates) in pancreatic carcinogenesis. However, very little is known about how the ISG15 pathway may contribute to pancreatic tumorigenesis. In the current study, we demonstrate that ISG15 pathway knockdown reverses the KRAS-associated phenotypes of PDAC cells such as increased proliferation and colony formation. Furthermore, clustered regularly interspaced short palindromic repeats (CRISPR)-mediated ISG15 knockdown decreased tumor programmed death ligand-1 (PDL-1) expression leading to increased number of CD8+ tumor-infiltrating lymphocytes and decreased pancreatic tumor growth. In addition, the syngeneic subcutaneous mouse model revealed that knocking down the ISG15 pathway significantly decreased the rate of tumor incidence and increased the survival rate. Interestingly, the ISG15 knockdown-mediated PDL-1 downregulation in pancreatic tumors increased the efficacy of anti-programmed cell death protein-1 (PD-1) treatment. ISG15 knockdown in combination with anti-PD-1 treatment synergistically increased the number of CD8+ tumor-infiltrating lymphocytes. Additionally, ISG15 knockdown alone significantly decreased the number of tumor-infiltrating regulatory T cells (Tregs) compared to wild type tumors treated with anti-PD-1 antibody. Overall, these findings suggest that strategies to target the ISG15 pathway by itself or in combination with immunotherapy may lead to improved survival for patients diagnosed with PDAC.
KeywordsImmunotherapy ISG15 Pancreatic cancer Tumor microenvironment
Clustered regularly interspaced short palindromic repeats
Interferon regulatory factor-1
Interferon-stimulated gene 15
Pancreatic ductal adenocarcinoma
Programmed cell death protein-1
Programmed death ligand-1
Regulatory T cells
Julian Burks would like to thank Dr. Jay Berzofsky at the National Cancer Institute-Vaccine Branch for providing lab space and support for part of the studies.
JB conceived and designed the research; JPS, JB, AF, and SL performed the experiments; JB analyzed the data; JPS, JB, AF, and SL interpreted the results of the experiments; JB prepared the figures; JB drafted the manuscript; JPS, JB, AF, and SL edited and revised the manuscript; all the authors approved the content of the final manuscript version.
This work was supported by the NIH T32CA00968 Grant, a Department of Medicine pilot award and The Ruesch Center for the Cure of Gastrointestinal Cancers, Georgetown University Medical Center. These studies were conducted in part at the Lombardi Comprehensive Cancer Center Histopathology and Tissue Shared resource funded by NIH CA051008.
Compliance with ethical standards
Conflict of interest
The authors have declared that no conflict of interest exists.
All applicable international, national, and/or institutional guidelines for the care and use of animals were followed. All procedures performed in the studies involving animals were in accordance with the ethical standards of the Georgetown University. The IACUC Protocol Number was 2016-1193 and the protocol approval date was 10/05/2017.
C57BL/6 mice were purchased from Charles River Laboratories (Maryland).
Cell line authentication
Murine pancreatic cancer cells, Panc02, which is syngeneic to C57BL/6 mice,  were a gift to Dr. Smith from Professor Corbett (Wayne State University, MI, USA). The Panc02 cell line was donated via the National Cancer Institute cell bank repository and the cell line was authenticated there. IMPACT-III testing was performed by IDEXX BioResearch (Columbia, MO, USA) to ensure the cells were pathogen-free.
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