Abstract
The immune checkpoint molecule, programmed death ligand 1 (PD-L1; B7-H1, CD274), induces T cell apoptosis and tolerance, thus inhibiting the antitumor immunity. PD-L1 expression is increased in many types of cancer, including ovarian cancer (OC), and correlates with poor prognosis. However, the mechanisms that regulate the PD-L1 expression in cancer cells are incompletely understood. The transcriptional regulation of PD-L1 expression is orchestrated by several transcription factors, including NFκB. The human PD-L1 promoter contains five NFκB-binding sites. Interferon-γ (IFNγ) stimulation of OC cells induces p65, and particularly K314/315 acetylated p65 recruitment to all five NFκB-binding sites in PD-L1 promoter, resulting in increased PD-L1 expression. In this chapter, we describe a protocol that uses chromatin immunoprecipitation (ChIP) to analyze the transcriptional regulation of PD-L1 by measuring recruitment of NFκB p65 and K314/315 acetylated p65 to PD-L1 promoter in human OC cells.
This is a preview of subscription content, log in via an institution.
Buying options
Tax calculation will be finalised at checkout
Purchases are for personal use only
Learn about institutional subscriptionsSpringer Nature is developing a new tool to find and evaluate Protocols. Learn more
References
Huang G, Wen Q, Zhao Y et al (2013) NF-κB plays a key role in inducing CD274 expression in human monocytes after lipopolysaccharide treatment. PLoS One 8:e61602
Peng J, Hamanishi J, Matsumura N et al (2015) Chemotherapy induces programmed cell death-ligand 1 overexpression via the nuclear factor-κB to Foster an immunosuppressive tumor microenvironment in ovarian cancer. Cancer Res 75:5034–5045
Gowrishankar K, Gunatilake D, Gallagher SJ et al (2015) Inducible but not constitutive expression of PD-L1 in human melanoma cells is dependent on activation of NF-κB. PLoS One 10:e0123410
Lim SO, Li CW, Xia W et al (2016) Deubiquitination and stabilization of PD-L1 by CSN5. Cancer Cell 30:925–939
Bouillez A, Rajabi H, Jin C et al (2017) MUC1-C integrates PD-L1 induction with repression of immune effectors in non-small-cell lung cancer. Oncogene 36:4037–4046
Maeda T, Hiraki M, Jin C et al (2018) MUC1-C induces PD-L1 and immune evasion in triple-negative breast cancer. Cancer Res 78:205–215
Abiko K, Matsumura N, Hamanishi J et al (2015) IFN-gamma from lymphocytes induces PD-L1 expression and promotes progression of ovarian cancer. Br J Cancer 112:1501–1509
Zou Y, Uddin MM, Padmanabhan S et al (2018) The proto-oncogene Bcl3 induces immune checkpoint PD-L1 expression, mediating proliferation of ovarian cancer cells. J Biol Chem 293:15483–15496
Acknowledgment
This work was supported by NIH grant CA202775 to I. Vancurova.
Author information
Authors and Affiliations
Corresponding author
Editor information
Editors and Affiliations
Rights and permissions
Copyright information
© 2020 Springer Science+Business Media, LLC, part of Springer Nature
About this protocol
Cite this protocol
Zou, Y., Padmanabhan, S., Vancurova, I. (2020). Analysis of PD-L1 Transcriptional Regulation in Ovarian Cancer Cells by Chromatin Immunoprecipitation. In: Vancurova, I., Zhu, Y. (eds) Immune Mediators in Cancer. Methods in Molecular Biology, vol 2108. Humana, New York, NY. https://doi.org/10.1007/978-1-0716-0247-8_20
Download citation
DOI: https://doi.org/10.1007/978-1-0716-0247-8_20
Published:
Publisher Name: Humana, New York, NY
Print ISBN: 978-1-0716-0246-1
Online ISBN: 978-1-0716-0247-8
eBook Packages: Springer Protocols