p62/SQSTM1-TRAF6/RIP1 complexes activate NF-κB-mediated PD-L1 expression and promote T-cell apoptosis in MKN45 gastric cancer cells.

Authors

Shiyang Li, Lingling Guo, Xuan Yang, Minwei Yang, Xiaoqian Li, Tao Ye, Min Jiang, Jian Ma

Published in

Journal of gastrointestinal oncology. Volume 17. Issue 3. Pages 136. Jun 30, 2026. Epub May 27, 2026.

Abstract

Autophagy has been implicated in the regulation of programmed death-ligand 1 (PD-L1) expression in gastric cancer (GC) through a p62/SQSTM1-nuclear factor-κB (NF-κB) pathway. However, the adaptor complexes linking p62 to NF-κB activation and the functional impact of this axis on T-cell apoptosis remain incompletely understood. This study aimed to investigate the role of p62/SQSTM1-TRAF6/RIP1 complexes in NF-κB-mediated PD-L1 expression and their effects on T-cell proliferation, apoptosis, and cytokine secretion in MKN-45 gastric cancer cells.
Human GC MKN45 cells were treated with the autophagy inhibitor chloroquine (CQ) and the NF-κB inhibitor, respectively. Following transfection with p62/SQSTM1 knockdown plasmids, cells were treated with CQ. PD-L1 and pathway-related molecules were assessed by real-time quantitative polymerase chain reaction (RT-qPCR), Western blotting and immunofluorescence. Protein-protein interactions were examined by co-immunoprecipitation. A co-culture system of MKN45 cells and activated human T cells was established to evaluate T-cell proliferation with Cell Counting Kit-8 (CCK-8), apoptosis with cleaved caspase-3 and cytokine production [interferon-γ (IFN-γ), interleukin-10 (IL-10) and tumour necrosis factor-α (TNF-α) by enzyme-linked immunosorbent assay (ELISA)].
CQ treatment in MKN45 cells resulted in autophagy blockade accompanied by p62/SQSTM1 accumulation, NF-κB activation and increased PD-L1 expression at both messenger ribonucleic acid (mRNA) and protein levels. Modulation of p62 expression confirmed that p62 was required for CQ-induced NF-κB activation and PD-L1 up-regulation. Co-immunoprecipitation experiments demonstrated that p62 formed complexes with TRAF6 and RIP1, and that these interactions were enhanced by p62 overexpression or CQ treatment but attenuated by p62 knockdown. Functionally, co-culture with CQ-treated or p62-overexpressing MKN45 cells suppressed T-cell proliferation, increased T-cell apoptosis and decreased IFN-γ, IL-10 and TNF-α secretion. These effects were partially reversed by p62 knockdown, NF-κB inhibition.
This study improves our molecular understanding of the p62/SQSTM1-NF-κB-PD-L1 signalling pathway by implicating TRAF6 and RIP1 as adaptor proteins in MKN45 GC cells. It also provides evidence that activation of this pathway promotes T-cell apoptosis and cytokine suppression. These results suggest that p62/NF-κB/PD-L1 signalling could be targeted to enhance anti-tumour immunity in GC.

PMID:
42434251
Bibliographic data and abstract were imported from PubMed on 11 Jul 2026.

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