Skip to main content

Advertisement

SpringerLink
Log in

ISG15 pathway knockdown reverses pancreatic cancer cell transformation and decreases murine pancreatic tumor growth via downregulation of PDL-1 expression

  • Original Article
  • Published:
Cancer Immunology, Immunotherapy Aims and scope Submit manuscript

Abstract

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.

This is a preview of subscription content, log in via an institution to check access.

Access this article

Log in via an institution

Price excludes VAT (USA)
Tax calculation will be finalised during checkout.

Instant access to the full article PDF.

Institutional subscriptions

Fig. 1
Fig. 2
Fig. 3
Fig. 4
Fig. 5
Fig. 6

Similar content being viewed by others

Abbreviations

CRISPR:

Clustered regularly interspaced short palindromic repeats

IRF-1:

Interferon regulatory factor-1

ISG15:

Interferon-stimulated gene 15

PDAC:

Pancreatic ductal adenocarcinoma

PD-1:

Programmed cell death protein-1

PDL-1:

Programmed death ligand-1

Tregs:

Regulatory T cells

References

  1. Ryan DP, Hong TS, Bardeesy N (2014) Pancreatic adenocarcinoma. N Engl J Med 371(11):1039–1049. https://doi.org/10.1056/NEJMra1404198

    Article  CAS  PubMed  Google Scholar 

  2. Desai SD, Haas AL, Wood LM, Tsai YC, Pestka S, Rubin EH, Saleem A, Nur EKA, Liu LF (2006) Elevated expression of ISG15 in tumor cells interferes with the ubiquitin/26S proteasome pathway. Can Res 66(2):921–928

    Article  CAS  Google Scholar 

  3. Li XY, Yan J, Sun J, Li C, Jiang JY, Wang JM, Meng XN, Liang JJ (1866) Wang HQ (2019) BAG3 deletion suppresses stem cell-like features of pancreatic ductal adenocarcinoma via translational suppression of ISG15. Biochim Biophys Acta 5:819–827. https://doi.org/10.1016/j.bbamcr.2019.02.008

    Article  CAS  Google Scholar 

  4. Zuo C, Sheng X, Ma M, Xia M, Ouyang L (2016) ISG15 in the tumorigenesis and treatment of cancer: an emerging role in malignancies of the digestive system. Oncotarget 7(45):74393–74409. https://doi.org/10.18632/oncotarget.11911

    Article  PubMed  PubMed Central  Google Scholar 

  5. D’Cunha J, Ramanujam S, Wagner RJ, Witt PL, Knight E Jr, Borden EC (1996) In vitro and in vivo secretion of human ISG15, an IFN-induced immunomodulatory cytokine. J Immunol 157(9):4100–4108

    PubMed  Google Scholar 

  6. Andersen JB, Hassel BA (2006) The interferon regulated ubiquitin-like protein, ISG15, in tumorigenesis: friend or foe? Cytokine Growth Factor Rev 17(6):411–421

    Article  CAS  Google Scholar 

  7. Burks J, Reed RE, Desai SD (2013) ISGylation governs the oncogenic function of Ki-Ras in breast cancer. Oncogene. https://doi.org/10.1038/onc.2012.633

    Article  PubMed  Google Scholar 

  8. Tsai YC, Pestka S, Wang LH, Runnels LW, Wan S, Lyu YL, Liu LF (2011) Interferon-beta signaling contributes to Ras transformation. PLoS One 6(8):e24291

    Article  CAS  Google Scholar 

  9. Hummer BT, Li XL, Hassel BA (2001) Role for p53 in gene induction by double-stranded RNA. J Virol 75(16):7774–7777

    Article  CAS  Google Scholar 

  10. Wood LM, Sankar S, Reed RE, Haas AL, Liu LF, McKinnon P, Desai SD (2011) A novel role for ATM in regulating proteasome-mediated protein degradation through suppression of the ISG15 conjugation pathway. PLoS One 6(1):e16422

    Article  CAS  Google Scholar 

  11. Tripathi MK, Chaudhuri G (2005) Down-regulation of UCRP and UBE2L6 in BRCA2 knocked-down human breast cells. Biochem Biophys Res Commun 328(1):43–48

    Article  CAS  Google Scholar 

  12. Desai SD, Reed RE, Burks J, Wood LM, Pullikuth AK, Haas AL, Liu LF, Breslin JW, Meiners S, Sankar S (2012) ISG15 disrupts cytoskeletal architecture and promotes motility in human breast cancer cells. Exp Biol Med (Maywood) 237(1):38–49

    Article  CAS  Google Scholar 

  13. Li C, Wang J, Zhang H, Zhu M, Chen F, Hu Y, Liu H, Zhu H (2014) Interferon-stimulated gene 15 (ISG15) is a trigger for tumorigenesis and metastasis of hepatocellular carcinoma. Oncotarget 5(18):8429–8441. https://doi.org/10.18632/oncotarget.2316

    Article  PubMed  PubMed Central  Google Scholar 

  14. Garcia-Diaz A, Shin DS, Moreno BH, Saco J, Escuin-Ordinas H, Rodriguez GA, Zaretsky JM, Sun L, Hugo W, Wang X, Parisi G, Saus CP, Torrejon DY, Graeber TG, Comin-Anduix B, Hu-Lieskovan S, Damoiseaux R, Lo RS, Ribas A (2017) Interferon receptor signaling pathways regulating PD-L1 and PD-L2 expression. Cell Rep 19(6):1189–1201. https://doi.org/10.1016/j.celrep.2017.04.031

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  15. Lee SJ, Jang BC, Lee SW, Yang YI, Suh SI, Park YM, Oh S, Shin JG, Yao S, Chen L, Choi IH (2006) Interferon regulatory factor-1 is prerequisite to the constitutive expression and IFN-gamma-induced upregulation of B7-H1 (CD274). FEBS Lett 580(3):755–762. https://doi.org/10.1016/j.febslet.2005.12.093

    Article  CAS  PubMed  Google Scholar 

  16. Li Y, Li F, Jiang F, Lv X, Zhang R, Lu A, Zhang G (2016) A mini-review for cancer immunotherapy: molecular understanding of PD-1/PD-L1 pathway and translational blockade of immune checkpoints. Int J Mol Sci 17:7. https://doi.org/10.3390/ijms17071151

    Article  CAS  Google Scholar 

  17. Yang YQ, Dong WJ, Yin XF, Xu YN, Yang Y, Wang JJ, Yuan SJ, Xiao J, DeLong JH, Chu L, Xu HN, Zhou XM, Wang RW, Fang L, Liu XY, Zhang KJ (2016) Interferon-related secretome from direct interaction between immune cells and tumor cells is required for upregulation of PD-L1 in tumor cells. Protein Cell 7(7):538–543. https://doi.org/10.1007/s13238-016-0281-6

    Article  PubMed  PubMed Central  Google Scholar 

  18. Lenschow DJ, Giannakopoulos NV, Gunn LJ, Johnston C, O’Guin AK, Schmidt RE, Levine B, Virgin HWt (2005) Identification of interferon-stimulated gene 15 as an antiviral molecule during Sindbis virus infection in vivo. J Virol 79(22):13974–13983. https://doi.org/10.1128/jvi.79.22.13974-13983.2005

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  19. Sainz B Jr, Martin B, Tatari M, Heeschen C, Guerra S (2014) ISG15 is a critical microenvironmental factor for pancreatic cancer stem cells. Can Res 74(24):7309–7320. https://doi.org/10.1158/0008-5472.Can-14-1354

    Article  CAS  Google Scholar 

  20. Hu Y, Chen W, Yan Z, Ma J, Zhu F, Huo J (2019) Prognostic value of PD-L1 expression in patients with pancreatic cancer: a PRISMA-compliant meta-analysis. Medicine 98(3):e14006. https://doi.org/10.1097/md.0000000000014006

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  21. Gao HL, Liu L, Qi ZH, Xu HX, Wang WQ, Wu CT, Zhang SR, Xu JZ, Ni QX, Yu XJ (2018) The clinicopathological and prognostic significance of PD-L1 expression in pancreatic cancer: a meta-analysis. Hepatobil Pancreat Dis Int 17(2):95–100. https://doi.org/10.1016/j.hbpd.2018.03.007

    Article  Google Scholar 

  22. Moon JW, Kong SK, Kim BS, Kim HJ, Lim H, Noh K, Kim Y, Choi JW, Lee JH, Kim YS (2017) IFNgamma induces PD-L1 overexpression by JAK2/STAT1/IRF-1 signaling in EBV-positive gastric carcinoma. Sci Rep 7(1):17810. https://doi.org/10.1038/s41598-017-18132-0

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  23. Qian J, Wang C, Wang B, Yang J, Wang Y, Luo F, Xu J, Zhao C, Liu R, Chu Y (2018) The IFN-gamma/PD-L1 axis between T cells and tumor microenvironment: hints for glioma anti-PD-1/PD-L1 therapy. J Neuroinflamm 15(1):290. https://doi.org/10.1186/s12974-018-1330-2

    Article  CAS  Google Scholar 

  24. Seifert AM, Zeng S, Zhang JQ, Kim TS, Cohen NA, Beckman MJ, Medina BD, Maltbaek JH, Loo JK, Crawley MH, Rossi F, Besmer P, Antonescu CR, DeMatteo RP (2017) PD-1/PD-L1 blockade enhances T-cell activity and antitumor efficacy of imatinib in gastrointestinal stromal tumors. Clin Cancer Res 23(2):454–465. https://doi.org/10.1158/1078-0432.Ccr-16-1163

    Article  CAS  PubMed  Google Scholar 

  25. Lanitis E, Dangaj D, Irving M, Coukos G (2017) Mechanisms regulating T-cell infiltration and activity in solid tumors. Ann Oncol 28(suppl_12):xii18–xii32. https://doi.org/10.1093/annonc/mdx238

    Article  CAS  PubMed  Google Scholar 

  26. D’Cunha J, Knight E Jr, Haas AL, Truitt RL, Borden EC (1996) Immunoregulatory properties of ISG15, an interferon-induced cytokine. Proc Natl Acad Sci USA 93(1):211–215

    Article  Google Scholar 

  27. Durfee LA, Huibregtse JM (2012) The ISG15 conjugation system. Methods Mol Boil (Clifton, NJ) 832:141–149. https://doi.org/10.1007/978-1-61779-474-2_9

    Article  CAS  Google Scholar 

  28. Burks J, Reed RE, Desai SD (2015) Free ISG15 triggers an antitumor immune response against breast cancer: a new perspective. Oncotarget 6(9):7221–7231. https://doi.org/10.18632/oncotarget.3372

    Article  PubMed  PubMed Central  Google Scholar 

  29. Chen RH, Du Y, Han P, Wang HB, Liang FY, Feng GK, Zhou AJ, Cai MY, Zhong Q, Zeng MS, Huang XM (2016) ISG15 predicts poor prognosis and promotes cancer stem cell phenotype in nasopharyngeal carcinoma. Oncotarget 7(13):16910–16922. https://doi.org/10.18632/oncotarget.7626

    Article  PubMed  PubMed Central  Google Scholar 

  30. Bazhin AV, von Ahn K, Fritz J, Werner J, Karakhanova S (2018) Interferon-alpha up-regulates the expression of PD-L1 molecules on immune cells through STAT3 and p38 signaling. Front Immunol 9:2129. https://doi.org/10.3389/fimmu.2018.02129

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  31. Morimoto Y, Kishida T, Kotani SI, Takayama K, Mazda O (2018) Interferon-beta signal may up-regulate PD-L1 expression through IRF9-dependent and independent pathways in lung cancer cells. Biochem Biophys Res Commun 507(1–4):330–336. https://doi.org/10.1016/j.bbrc.2018.11.035

    Article  CAS  PubMed  Google Scholar 

  32. Duarte CW, Willey CD, Zhi D, Cui X, Harris JJ, Vaughan LK, Mehta T, McCubrey RO, Khodarev NN, Weichselbaum RR, Gillespie GY (2012) Expression signature of IFN/STAT1 signaling genes predicts poor survival outcome in glioblastoma multiforme in a subtype-specific manner. PLoS One 7(1):e29653. https://doi.org/10.1371/journal.pone.0029653

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  33. Que Y, Xiao W, Guan YX, Liang Y, Yan SM, Chen HY, Li QQ, Xu BS, Zhou ZW, Zhang X (2017) PD-L1 expression is associated with FOXP3+ regulatory T-cell infiltration of soft tissue sarcoma and poor patient prognosis. J Cancer 8(11):2018–2025. https://doi.org/10.7150/jca.18683

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  34. DiDomenico J, Lamano JB, Oyon D, Li Y, Veliceasa D, Kaur G, Ampie L, Choy W, Lamano JB, Bloch O (2018) The immune checkpoint protein PD-L1 induces and maintains regulatory T cells in glioblastoma. Oncoimmunology 7(7):e1448329. https://doi.org/10.1080/2162402x.2018.1448329

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  35. Francesca Romana Spinelli IP, Piconese Silvia, Ceccarelli Fulvia, Miranda Francesca, Alessandri Cristiano, Barnaba Vincenzo, Valesini Guido, Conti Fabrizio (2019) Interferon stimulated gene 15 protects regulatory T cell of systemic lupus erythematosus patients from interferon alpha-mediated depletion. Ann Rheum Dis 78:379–380

    Google Scholar 

  36. Corbett TH, Roberts BJ, Leopold WR, Peckham JC, Wilkoff LJ, Griswold DP Jr, Schabel FM Jr (1984) Induction and chemotherapeutic response of two transplantable ductal adenocarcinomas of the pancreas in C57BL/6 mice. Can Res 44(2):717–726

    CAS  Google Scholar 

Download references

Acknowledgements

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.

Funding

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.

Author information

Authors and Affiliations

  1. Department of Medicine, Georgetown University, Washington, DC, 20007, USA

    Julian Burks & Jill P. Smith

  2. Department of Oncology, Georgetown University, Washington, DC, 20007, USA

    Alia Fleury & Sarah Livingston

  3. Vaccine Branch, Center for Cancer Research, National Cancer Institute, Building 41, Room D702, Bethesda, MD, 20892, USA

    Julian Burks

Authors
  1. Julian Burks
    View author publications

    You can also search for this author in PubMed Google Scholar

  2. Alia Fleury
    View author publications

    You can also search for this author in PubMed Google Scholar

  3. Sarah Livingston
    View author publications

    You can also search for this author in PubMed Google Scholar

  4. Jill P. Smith
    View author publications

    You can also search for this author in PubMed Google Scholar

Contributions

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.

Corresponding author

Correspondence to Julian Burks.

Ethics declarations

Conflict of interest

The authors have declared that no conflict of interest exists.

Ethical standards

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.

Animal source

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, [36] 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.

Additional information

Publisher's Note

Springer Nature remains neutral with regard to jurisdictional claims in published maps and institutional affiliations.

Electronic supplementary material

Below is the link to the electronic supplementary material.

Supplementary material 1 (PDF 525 kb)

Rights and permissions

Reprints and permissions

About this article

Check for updates. Verify currency and authenticity via CrossMark

Cite this article

Burks, J., Fleury, A., Livingston, S. et al. ISG15 pathway knockdown reverses pancreatic cancer cell transformation and decreases murine pancreatic tumor growth via downregulation of PDL-1 expression. Cancer Immunol Immunother 68, 2029–2039 (2019). https://doi.org/10.1007/s00262-019-02422-9

Download citation

  • Received:

  • Accepted:

  • Published:

  • Issue Date:

  • DOI: https://doi.org/10.1007/s00262-019-02422-9

Keywords

Search

Navigation