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Intrinsic and Extrinsic Regulation of PD-L2 Expression in Oncogene-Driven Non-Small Cell Lung Cancer
Daisuke Shibahara1, Kentaro Tanaka2, Eiji Iwama2
1Research Institute for Diseases of the Chest, Graduate School of Medical Sciences, Kyushu University, Fukuoka, Japan; Department of Infectious Disease, Respiratory, and Digestive Medicine, Graduate School of Medicine, University of Ryukyus, Okinawa, Japan.
Introduction:
The interaction of programmed cell death ligand 2 (PD-L2) with programmed cell death 1 is implicated in tumor immune escape. The regulation of PD-L2 expression in tumor cells has remained unclear, however. We here examined intrinsic and extrinsic regulation of PD-L2 expression in NSCLC.
Methods:
PD-L2 expression was evaluated by reverse transcription and real-time polymerase chain reaction analysis and by flow cytometry.
Results:
BEAS-2B cells stably expressing an activated mutant form of EGFR or the echinoderm microtubule associated protein like 4 (EML4)-ALK receptor tyrosine kinase fusion oncoprotein manifested increased expression of PD-L2 at both the mRNA and protein levels. Furthermore, treatment of NSCLC cell lines that harbor such driver oncogenes with corresponding EGFR or ALK tyrosine kinase inhibitors or depletion of EGFR or ALK by small interfering RNA transfection suppressed expression of PD-L2, demonstrating that activating EGFR mutations or echinoderm microtubule associated protein like 4 gene (EML4)-ALK receptor tyrosine kinase gene (ALK) fusion intrinsically induce PD-L2 expression. We also found that interferon gamma (IFN-γ) extrinsically induced expression of PD-L2 through signal transducer and activator of transcription 1 signaling in NSCLC cells. Oncogene-driven expression of PD-L2 in NSCLC cells was inhibited by knockdown of the transcription factors signal transducer and activator of transcription 3 (STAT3) or c-FOS. IFN-γ also activated STAT3 and c-FOS, suggesting that these proteins may also contribute to the extrinsic induction of PD-L2 expression.
Conclusions:
Expression of PD-L2 is induced intrinsically by activating EGFR mutations or EML4-ALK fusion and extrinsically by IFN-γ, with STAT3 and c-FOS possibly contributing to both intrinsic and extrinsic pathways. Our results thus provide insight into the complexity of tumor immune escape in NSCLC.
Insights
Programmed cell death ligand 2 (PD-L2) expression in non-small cell lung cancer (NSCLC) is driven by EGFR mutations or EML4-ALK fusions and interferon-gamma (IFN-γ). STAT3 and c-FOS transcription factors play a role in PD-L2 regulation.
Area of Science:
- Oncology
- Immunology
- Molecular Biology
Background:
- Programmed cell death ligand 2 (PD-L2) interaction with programmed cell death 1 (PD-1) is crucial in tumor immune evasion.
- The precise regulation of PD-L2 expression within tumor cells remains incompletely understood.
- Investigating PD-L2 regulation is key to understanding and potentially overcoming tumor immune escape mechanisms in non-small cell lung cancer (NSCLC).
Purpose of the Study:
- To elucidate the intrinsic and extrinsic factors regulating PD-L2 expression in NSCLC.
- To determine the role of specific oncogenic drivers (EGFR, EML4-ALK) in PD-L2 upregulation.
- To explore the influence of external factors like interferon-gamma (IFN-γ) on PD-L2 expression.
Main Methods:
- Quantitative analysis of PD-L2 mRNA and protein levels using reverse transcription and real-time polymerase chain reaction (RT-PCR) and flow cytometry.
- Utilizing NSCLC cell lines with specific driver oncogenes (EGFR, EML4-ALK).
- Employing tyrosine kinase inhibitors (TKIs), small interfering RNA (siRNA) for gene depletion, and stimulation with IFN-γ.
Main Results:
- Activating mutations in EGFR or EML4-ALK fusions significantly increased PD-L2 mRNA and protein expression in NSCLC cells.
- Inhibition of EGFR or ALK, or depletion of these targets, suppressed PD-L2 expression, confirming intrinsic oncogene-driven regulation.
- Interferon-gamma (IFN-γ) was identified as an extrinsic inducer of PD-L2 expression via Signal Transducer and Activator of Transcription 1 (STAT1) signaling.
- Knockdown of STAT3 or c-FOS inhibited oncogene-driven PD-L2 expression, and IFN-γ also activated these factors, suggesting their involvement in both intrinsic and extrinsic pathways.
Conclusions:
- PD-L2 expression in NSCLC is intrinsically regulated by oncogenic drivers like EGFR mutations and EML4-ALK fusions.
- Extrinsic induction of PD-L2 by IFN-γ involves STAT1 signaling and potentially STAT3 and c-FOS.
- STAT3 and c-FOS may act as convergent points for both intrinsic and extrinsic PD-L2 regulation, highlighting the complex interplay in tumor immune escape.
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