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

Introduction

Gastric cancer (GC) is one of the most prevalent malignant tumours worldwide, with consistent high incidence and mortality rates (1). In China in particular, the disease burden is exceptionally high, accounting for almost half of all new cases and deaths worldwide each year (2). Despite advances in surgical techniques and the adoption of comprehensive treatment regimens that improve diagnostic and therapeutic outcomes, the prognosis for patients with advanced or metastatic GC remains poor. In recent years, tumour immunotherapy, particularly immune checkpoint inhibitor (ICI) treatments targeting programmed death-1 (PD-1) and programmed death-ligand 1 (PD-L1), has made significant progress in treating multiple solid tumours (3). However, in GC treatment, the efficacy of ICIs varies significantly from person to person. Clinical data indicate that only a subset of GC patients benefits from anti-PD-1/PD-L1 therapy, with overall response rates ranging from 10% to 26% (4). This prevalent phenomenon of primary or secondary resistance severely limits the efficacy of immunotherapy in GC, representing a critical bottleneck in current treatment approaches.

In GC, one of the most critical scientific challenges is to delve into the mechanisms underlying the formation of the immunosuppressive microenvironment, particularly the intricate regulation of PD-L1 expression on tumour cells, in order to overcome immune therapy resistance and enhance treatment efficacy. PD-L1 is expressed on the surface of tumour cells and immune cells. It inhibits T cell activation, proliferation, and cytokine secretion by binding to PD-1 on the surface of T cells (5). PD-L1 upregulation is a key mechanism by which tumour cells evade immune surveillance. The expression of PD-L1 in cancer cells is influenced by multiple intrinsic and extrinsic factors, including oncogenic signalling pathways, inflammatory cytokines and the tumour microenvironment. Traditional understanding has largely focused on the transcriptional induction of PD-L1 by inflammatory signals [interferon-γ (IFN-γ)]. However, in the unique tumour microenvironment of GC, particularly under conditions such as chronic inflammation and abnormal autophagy, the regulatory network governing PD-L1 expression becomes significantly more complex (6,7). In cell signalling pathways, nuclear factor-κB (NF-κB) acts as a main regulator of inflammatory and immune responses, and its involvement in the transcriptional regulation of PD-L1 has been demonstrated across multiple tumour types (8). Nevertheless, the molecular events that link cellular stress pathways to NF-κB-dependent PD-L1 expression in GC are not fully understood.

Autophagy is a vital intracellular catabolic process that degrades damaged organelles and misfolded proteins, thereby maintaining cellular homeostasis (9). P62/SQSTM1 is a selective autophagy adaptor protein that recognises and transports ubiquitinated proteins to autophagosomes. When autophagy flow is impaired, p62 accumulates and acts as a signaling hub regulating multiple pathways including NF-κB, MAPK, and mTOR. Previous studies indicate that p62 promotes K63-linked ubiquitination and NF-κB activation in diverse cellular environments by interacting with adaptor proteins such as tumour necrosis factor receptor-associated factor 6 (TRAF6) and receptor-interacting protein 1 (RIP1) (10,11). These findings suggest that autophagy-dependent regulation of p62 may influence immune checkpoint signaling in cancer cells. In GC, previous studies have demonstrated that autophagy influences PD-L1 expression. Specifically, Wang et al. confirmed that pharmacological or genetic inhibition of autophagy enhances PD-L1 expression in GC cells and mouse models (7), with this effect involving p62/SQSTM1 accumulation and NF-κB activation. This study established the functional linkage between autophagy, p62, and NF-κB in regulating PD-L1 expression in GC. However, the upstream adaptor complex linking p62 to NF-κB activation remains incompletely elucidated, and the impact of the autophagy-p62-NF-κB-PD-L1 on T-cell apoptosis and effector function has not been systematically investigated. Consequently, the detailed molecular mechanisms of p62-mediated NF-κB activation in GC and its immunological consequences require further clarification.

This study employed the human GC cell line MKN45 as a model to further elucidate the p62/SQSTM1-NF-κB-PD-L1. We first demonstrated that chloroquine (CQ) inhibition of autophagy leads to p62 accumulation, NF-κB activation, and PD-L1 upregulation in MKN45 cells, with p62 being indispensable in this process. Subsequently, we investigated whether p62 forms complexes with TRAF6 and RIP1, and how these interactions correlate with NF-κB activation. Finally, using a co-culture system of MKN45 cells with activated human T cells, we assessed the functional impact of this pathway on T cell proliferation, apoptosis, and cytokine production, while analyzing the effects of targeting p62, NF-κB, and PD-L1. Our findings enhance molecular understanding of autophagy-related PD-L1 regulation and provide functional evidence that activation of the p62/SQSTM1-NF-κB-PD-L1 promotes T cell apoptosis and immune evasion in GC cells. We present this article in accordance with the MDAR reporting checklist (available at https://jgo.amegroups.com/article/view/10.21037/jgo-2026-1-0106/rc).

Methods

Cell culture

The human GC cell lines MKN-45 and HGC27, as well as the normal gastric epithelial cell line GES-1, were purchased from Sunncell (Wuhan, China). All cells were cultured in low-glucose RPMI-1640 complete medium supplemented with 10% fetal bovine serum and 1% penicillin-streptomycin solution. Cells were maintained in the logarithmic growth phase and routinely passaged for subsequent experiments.

Cell grouping

Control group (NC): MKN-45 cells cultured normally without any treatment. Autophagy inhibition group (CQ): treated with CQ for 24 hours. Knockdown combined autophagy inhibition group (Si-p62 + CQ): first transfected with si-p62, then treated with CQ for 24 hours. Combined inhibition group (BAY + CQ): co-treated with BAY 11-7082 and CQ for 24 hours. Autophagy combined with negative control antibody group (CQ + anti-lgG): after 24 hours of treatment with CQ, 20 µg/µL negative control antibody was added. Autophagy combined with negative control antibody group (CQ + anti-PD-L1): after 24 hours of treatment with CQ, 20 µg/µL PD-L1 monoclonal antibody was added.

Cell transfection

The cells were cultured overnight in a 24-well plate. When the cell density reached about 60−70%, si-p62 were transfected into the cells according to the instructions of Lipofectamine 3000 reagent (Invitrogen, Grand Island, NY, USA). The cells were cultured at 37 °C and 5% CO₂ incubator for 48 h. Transfection efficiency was verified by real-time quantitative polymerase chain reaction (RT-qPCR) at 48 hours post-transfection. The knockdown efficiency of p62 was confirmed by significantly decreased messenger ribonucleic acid (mRNA) and protein expression of p62 in the si-p62 group.

Cell Counting Kit-8 (CCK-8) assay

The CCK-8 test kit (Beyotime Biotechnology, Beijing, China) was used to detect cell viability. Each group of cells were added to a 96-well plate at a concentration of 3×10³ cells per well. Subsequently, 10 µL of CCK-8 solution was introduced and incubated at 37 °C for 1–3 h. An enzyme labelling instrument was utilised to measure the absorbance values at 450 nm.

RT-qPCR

The total RNA of the cells were extracted with RNA Easy FAST Kit (Tiangen Biotech, Beijing, China) and reverse transcribed into single-stranded complementary DNA (cDNA) by FastKing-RT SuperMix Kit (Tiangen Biotech, Beijing, China). Using Applied Biosystems^TMPowerUp^TM SYBRT^M Green (Thermo, Waltham, USA), follow the manufacturer’s procedures for RT-qPCR. The sequence of RT-qPCR primers is shown in Table 1. Taking GAPDH as the internal reference, the value was calculated by the 2^−ΔΔCt method.

Western blotting analysis

Total proteins from cells were extracted using RIPA lysis buffer supplemented with a protease inhibitor cocktail. The protein concentration of each sample was determined via the BCA assay. Equal amounts of protein (20 μg) were resolved by sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) and subsequently transferred to polyvinylidene fluoride (PVDF) membranes. The membranes were then blocked with a rapid blocking buffer, followed by incubation with primary antibodies overnight at 4 °C and appropriate secondary antibodies at room temperature. Protein bands were detected using an enhanced chemiluminescence (ECL) chemiluminescence kit, and band intensities were quantified with ImageJ software. The following antibodies were used: anti-GAPDH (Abcam, Cambridge, United Kingdom, ab181602, 1:1,000), anti-β-actin (Merck, Darmstadt, Germany, SAB3500350, 1:1,000), anti-p62 (Abcam, ab109012, 1:1,000), anti-PD-L1 (Abcam, ab205921, 1:1,000), anti-IκBα (Merck, SAB1305978, 1:1,000), antip-IκBα (Merck, ZRB1554, 1:1,000), anti-p-p65 (Merck, SAB4504490, 1:1,000).

Co-immunoprecipitation (Co-IP)

Protein–protein interactions between p62 and NF-κB adaptor proteins were analyzed by Co-IP. MKN-45 cells subjected to the indicated treatments were lysed in a non-denaturing IP buffer containing protease inhibitors. Lysates were pre-cleared with protein A/G agarose beads for 1 h at 4 °C and then incubated overnight at 4 °C with antibodies against p62, TRAF6 or RIP1, or with isotype-matched IgG as negative controls. Immune complexes were captured by incubation with fresh protein A/G Sepharose (MCE) for 2–4 h at 4 °C and washed extensively with cold IP buffer. Bound proteins were eluted by boiling the beads in SDS sample buffer and analyzed by Western blotting with antibodies against TRAF6 (Merck, SAB3500377) and RIP1 (Merck, ZRB1767).

ChIP-qPCR

ChIP assays were performed to evaluate the binding of NF-κB p65 to the PD-L1 promoter. MKN-45 cells were cross-linked with 1% formaldehyde at room temperature for 10 minutes. The cross-linking reaction was quenched with a glycine solution at a concentration of 125 mM. The cells were then washed with cold PBS, harvested and lysed, and the chromatin sheared by sonication into fragments approximately 200–500 bp in size. The sheared chromatin was incubated overnight at 4 °C with either an anti-p65 antibody or a control IgG, together with protein A/G beads. Following extensive washing, the immune complexes were eluted and the cross-links were reversed by incubation at 65 °C. The DNA was then purified using a spin column kit and subjected to qPCR using primers spanning the predicted NF-κB binding sites in the PD-L1 promoter region. The ChIP-qPCR data were normalised to input DNA and expressed as a fold enrichment relative to the IgG controls.

Immunofluorescence staining

For immunofluorescence analysis, MKN-45 cellswere seeded on sterile glass coverslips in 24-well plates and subjected to the indicated treatments. Cells were fixed with 4% paraformaldehyde for 15–20 min, washed with PBS and permeabilized with 0.1–0.3% Triton X-100 for 10–15 min where appropriate. After blocking in 5% bovine serum albumin (BSA) for 1 h at room temperature, cells were incubated with primary antibodies against p62, TRAF6 and RIP1 at 4 °C overnight. After washing, the cells were incubated with species-appropriate, fluorophore-conjugated secondary antibodies for 1 hour at room temperature and in the dark. The nuclei were then counterstained with DAPI for 5–10 minutes. The slides were then mounted with an antifade medium and examined using a fluorescence microscope to check for co-localisation of p62 with TRAF6/RIP1.

Isolation and activation of CD8+ T cells

Peripheral blood mononuclear cells (PBMCs) were isolated with Ficoll-Paque using density gradient centrifugation. The CD8⁺ T cells were then purified from the PBMCs using a magnetic bead-based isolation kit according to the manufacturer’s protocol. The purified CD8⁺ T cells were cultured in RPMI-1640 medium supplemented with 10% FBS, and activated with plate-bound anti-CD3 and soluble anti-CD28 antibodies in the presence of recombinant human IL-2 for 24–48 hours. The activated CD8⁺ T cells were then used for co-culture experiments.

Cell co-culture

To assess the functional impact of the p62/NF-κB/PD-L1 on CD8⁺ T cells, MKN-45 cells were seeded in 24-well plates. After pretreatment, activated CD8⁺ T cells were added to MKN-45. Co-cultures were maintained in complete medium for 24–48 h. After co-culture, supernatants were collected for cytokine analysis, and T cells were harvested for proliferation and apoptosis assays.

Enzyme-linked immunosorbent assay (ELISA)

The concentrations of IFN-γ, tumour necrosis factor-α (TNF-α), and interleukin-10 (IL-10) in the co-culture supernatants of MKN-45-CD8+ T cells are to be determined using an ELISA kit, in accordance with the manufacturer’s instructions. Supernatants were centrifuged to remove cell debris and stored at −80 °C until analysis. Standards and samples were added to antibody-coated plates and incubated as recommended. After sequential incubation with detection antibodies and substrate solution, absorbance at 450 nm was measured. Standard curves were established in parallel, and cytokine concentrations were calculated according to the standard curves. All samples were tested in triplicate to ensure reliability.

T-cell apoptosis assay

T-cell apoptosis in co-culture experiments was evaluated by detection of cleaved caspase-3. The concentrations of cleaved caspase-3 in the co-culture of MKN-45-CD8+ T cells are to be determined using an cleaved caspase-3 kit, in accordance with the manufacturer's instructions. The absorbance was detected at the corresponding wavelength, and the relative apoptosis level was calculated to reflect the apoptotic degree of CD8⁺ T cells in each group.

Statistical analysis

GraphPad Prism 9.0 (GraphPad Software, Inc., La Jolla, CA, USA) was used for statistical analyses. All data are given as the (mean ± standard deviation). Statistical analyses were performed using a two-tailed Student’s t-test for two-group comparisons, one-way analysis of variance (ANOVA) for multi group comparisons, and a Kaplan-Meier nonparametric log-rank test for survival rate comparisons. All in vitro experiments were performed with at least three independent biological replicates, and each biological replicate included three technical replicates. Statistical analyses were performed based on biological replicates. P≤0.05 was considered significant (*, P<0.05; **, P<0.01; ***, P<0.001; and ns, nonsignificant).

Results

p62 accumulation is required for CQ-induced PD-L1 upregulation

RT-PCR and Western blot analyses were first performed to compare p62 expression between the GC cell line MKN-45, HGC27 and the normal gastric epithelial cell line GES-1. Both p62 mRNA and protein levels were markedly lower in MKN-45 than in GES-1. Compared with the HGC27 cell line, p62 expression was markedly higher in MKN45 cells, further validating the representativeness of MKN45 cells in this study (Figure 1A). To investigate the role of p62, MKN-45 were transfected with si-p62 or NC. RT-PCR confirmed efficient knockdown of p62 in the si-p62 group compared with the NC group (Figure 1B). CCK-8 assays demonstrated that treatment with the autophagy inhibitor CQ significantly enhanced the proliferative capacity of MKN-45 compared to the control, whereas the proliferation of the si-p62 + CQ group did not differ significantly from that of the NC group (Figure 1C). RT-PCR and Western blotting further revealed that CQ treatment led to a pronounced accumulation of p62, accompanied by a significant increase in PD-L1 mRNA and protein expression (Figure 1D,1E). Notably, in si-p62, CQ failed to induce comparable PD-L1 upregulation; the CQ increase in PD-L1 expression was markedly decreased in the si-p62 group (Figure 1E). These results indicate that p62 accumulation is necessary for CQ-induced PD-L1 upregulation in MKN-45.

Figure 1 p62 accumulation is required for CQ-induced PD-L1 upregulation in gastric cancer cells. (A) The mRNA and protein expression levels of p62 in gastric cancer cell lines (MKN-45, HGC27) and normal gastric epithelial cell line GES-1 were detected by RT-qPCR and Western blotting. (B) The knockdown efficiency of si-p62 in MKN-45 cells was verified by RT-qPCR. (C) Cell proliferative activity in the NC, CQ, and si-p62 + CQ groups was measured using CCK-8 assay. (D) The mRNA and protein expression of p62 in MKN-45 cells under different treatments were determined by RT-qPCR and Western blotting. (E) The mRNA and protein levels of PD-L1 in each group were detected by RT-qPCR and Western blotting. All data are presented as mean ± SD of at least three independent experiments. ns, nonsignificant; *, P<0.05; **, P<0.01; ***, P<0.001. CCK-8, Cell Counting Kit-8; CQ, chloroquine; PD-L1, programmed death-ligand 1; RT-qPCR, real-time quantitative polymerase chain reaction; SD, standard deviation.

p62 upregulates PD-L1 through activation of the NF-κB pathway

To further clarify how p62 regulates PD‑L1 expression, we focused on the NFκB signaling pathway. Based on previous report (12), BAY 11‑7082 was selected as a pharmacological NF‑κB inhibitor. MKN‑45 were treated with different concentrations of BAY 11‑7082, and cell viability was assessed by CCK‑8 after 48 h. A concentration of 10 µM BAY 11‑7082 produced the most pronounced inhibitory effect on cell proliferation. Western blot analysis confirmed effective inhibition of NF‑κB signaling at this concentration (Figure 2A). We then treated MKN‑45 with CQ alone or in combination with BAY 11‑7082. CCK‑8 assays showed that the enhanced proliferative capacity observed in the CQ group was decreased when BAY 11‑7082 was added; the proliferation of the CQ + BAY group was not significantly different from that of the control group (Figure 2B). qRT‑PCR and Western blotting demonstrated that CQ treatment significantly increased PD‑L1 mRNA and protein levels, whereas co‑treatment with BAY 11‑7082 markedly suppressed this CQ‑induced PD‑L1 up‑regulation (Figure 2C,2D). As expected, BAY 11‑7082 did not affect p62 accumulation induced by CQ, indicating that the inhibitor acts downstream of p62. At the signaling level, CQ treatment led to an increase in p-IκBα and an accumulation of p65 in the nucleus, indicating activation of the NF‑κB pathway (Figure 2E). Furthermore, CQ appeared to significantly activate the WT PD-L1 promoter, while CQ did not activate the PD-L1 promoter at the mutated NF-κB site; at the same time, knocking down p62 attenuated the activation of the WT promoter (Figure 2F). The results indicate that PD-L1 upregulation is largely dependent on the NF‑κB binding site, and p62 may act as an upstream regulatory factor for this transcriptional event. These changes were reversed by BAY 11‑7082. Taken together, these findings support the notion that p62 regulates PD‑L1 expression in MKN‑45 in a manner dependent on NF‑κB pathway activation.

Figure 2 Regulation of PD-L1 by p62 by the NF-κB pathway. (A) The inhibitory effect of BAY 11-7082 on cell proliferation and NFκB signaling was evaluated by CCK-8 and Western blotting. (B) Cell proliferative capacity in the NC, CQ, and CQ + BAY groups was detected using CCK-8 assay. (C,D) The mRNA and protein expression levels of p62 and PD-L1 in each group were measured by RTqPCR and Western blotting. (E) The protein levels of p-IκBα, IκBα, and p-p65 related to NF-κB signaling were detected by Western blotting. (F) PD-L1 promoter luciferase reporter assay showing the transcriptional activity of wild-type (WT) and NF-κB-binding-site-mutated (MUT) PD-L1 promoters in the NC, CQ, and CQ + si-p62 groups. All data are presented as mean ± SD from three independent experiments. ns, nonsignificant; **, P<0.01; ***, P<0.001 versus the NC group; ^##, P<0.01 versus the CQ group. CCK-8, Cell Counting Kit8; CQ, chloroquine; MUT, mutated; PD-L1, programmed death-ligand 1; RT-qPCR, real-time quantitative polymerase chain reaction; SD, standard deviation; WT, wild-type.

Direct molecular interactions between p62 and the NF‑κB pathway

To further explore the mechanistic relationship between p62 and NF‑κB signaling, we first performed ChIPqPCR targeting the PD‑L1 promoter. The results showed that p62 overexpression enhanced the binding of p65 to the PD‑L1 promoter region, which may contribute to PD‑L1 transcription (Figure 3A). This implies that p62 may play a role in the NF-κB-mediated transcriptional activation of PD-L1. Next, we examined the interaction between p62 and upstream NF-κB adaptor proteins using Co-IP. These experiments revealed that p62 physically associates with TRAF6 and RIP1 in MKN-45 cells. In the CQ (autophagy-inhibited) group, the interaction between p62 and TRAF6/RIP1 was markedly enhanced compared to the control group. In contrast, only weak binding was detected in the control group (Figure 3B,3C). When the relevant functional domain of p62 was mutated, its binding to TRAF6 was significantly reduced, supporting the potential importance of this domain in mediating the interaction and signal transduction.

Figure 3 p62 physically interacts with TRAF6/RIP1 to activate NF-κB signaling in MKN-45 cells. (A) ChIP-qPCR analysis showing the enrichment of NF-κB p65 on the PD-L1 promoter in control and p62-overexpressing MKN-45 cells. (B,C) Co-IP assays showing the endogenous interactions between p62 and TRAF6, and between p62 and RIP1 under normal and CQ-treated conditions. (D) Immunofluorescence staining showing the colocalization of p62 with TRAF6 and RIP1 in MKN-45 cells with or without CQ treatment. Original magnification, ×400. *, P<0.05; **, P<0.01; ***, P<0.001. Co-IP, co-immunoprecipitation; CQ, chloroquine; PD-L1, programmed death-ligand 1.

The intracellular localisation of p62, TRAF6 and RIP1 was visualised using immunofluorescence staining. The results showed increased co-localisation of p62 with TRAF6 and RIP1 in CQ group compared with control group (Figure 3D). Collectively, these data are consistent with the idea that p62 accumulation enhances its interaction and co-localisation with the NF-κB pathway, supporting a direct molecular link between p62 and NF-κB signaling in the regulation of PD-L1 expression.

Functional role of the p62/NF‑κB/PD‑L1 in tumour immune escape

Having established a potential molecular interaction between p62 and the NF-κB pathway, we investigated the possible functional consequences of the p62/NF-κB/PDL1 axis on antitumour immunity. MKN-45 subjected to various treatments were co-cultured with activated CD8⁺ T cells. We measured the levels of T-cell cytokines in the culture medium using ELISA. Compared with the CQ + BAY group, the CQ group exhibited significantly lower concentrations of IFN-γ and IL-10, while TNF-α levels were higher (Figure 4A). CCK-8 assays also revealed that CD8⁺ T-cell proliferation was markedly reduced in the CQ group compared to the control group. However, the addition of BAY 11-7082 partially restored T-cell proliferative capacity (Figure 4B). Assessment of T-cell apoptosis showed that the rate of apoptosis in CD8⁺ T cells was significantly higher in the CQ group but lower when NF-κB signalling was inhibited by BAY 11-7082 (Figure 4C). Furthermore, in the CQ + anti-PD-L1 group, the secretion levels of IFN-γ and IL-10 were markedly elevated relative to the CQ + anti-IgG group, whereas TNF-α expression was decreased (Figure 5A). In comparison with the CQ + anti‑IgG group, CD8⁺ T cells in the CQ + anti‑PD‑L1 group exhibited obviously enhanced proliferative capacity and a notably reduced apoptosis rate. In addition, pharmacological inhibition of NF-κB, as well as blockade with PD‑L1 monoclonal antibody, appeared to effectively reverse the CQ-induced immunosuppressive phenotype (Figure 5B,5C). These results provide evidence supporting that activation of the p62/NF-κB/PD-L1 in MKN-45 may contribute to T-cell dysfunction characterised by impaired proliferation, altered cytokine secretion and increased apoptosis. Collectively, these findings are consistent with the idea that the p62/NF-κB/PD-L1 axis may participate in mediating GC immune escape.

Figure 4 The p62/NF-κB axis mediates immunosuppression in gastric cancer cells. MKN-45 cells were treated as indicated and co-cultured with activated CD8⁺ T cells. (A) The concentrations of IFN-γ, TNF-α, and IL-10 in the culture supernatants were measured by ELISA. (B) CD8⁺ T-cell proliferation was evaluated using CCK-8 assay. (C) CD8⁺ T-cell apoptosis was assessed by detecting the relative level of cleaved caspase-3. All data are expressed as mean ± SD of three independent experiments. *, P<0.05; **, P<0.01; ***, P<0.001. CCK-8, Cell Counting Kit-8; SD, standard deviation.

Figure 5 The p62/NF-κB/PD-L1 axis promotes CD8⁺ T-cell dysfunction and immune escape in gastric cancer. MKN-45 cells were pretreated with CQ, CQ + BAY 11-7082, CQ + anti-IgG, or CQ + anti-PD-L1, then co-cultured with activated CD8⁺ T cells. (A) The secretion levels of IFN-γ, TNF-α, and IL-10 in the supernatants were determined by ELISA. (B) CD8⁺ T-cell proliferation was detected by CCK-8 assay. (C) CD8⁺ T-cell apoptosis was measured by quantifying cleaved caspase-3. *, P<0.05; **, P<0.01. CCK-8, Cell Counting Kit-8; CQ, chloroquine; PD-L1, programmed death-ligand 1.

Discussion

As a core process for maintaining cellular homeostasis, autophagy is closely linked to tumourigenesis and progression, particularly through its increasingly recognised role in shaping the tumour immune microenvironment. Accumulating evidence has demonstrated that autophagy serves as a critical regulator of PD-L1 expression in various cancers, including GC. Notably, Wang et al. (7) first reported that autophagy inhibition enhances PD-L1 expression via p62/SQSTM1 accumulation and subsequent NF-κB activation in GC cells, establishing a fundamental link between autophagy, p62, and PD-L1. However, the upstream adaptor complexes that connect p62 to NF‑κB signalling, as well as the direct functional impact of this axis on CD8⁺ T-cell apoptosis and dysfunction, remained poorly defined. Therefore, the present study was designed to further clarify the molecular mechanism by which p62 regulates PD-L1 transcription and modulates anti‑tumour immunity. Our data confirm that p62 accumulation induced by autophagy inhibition is required for PD-L1 upregulation through NF-κB signalling, and we further identify TRAF6/RIP1 as key upstream adaptors that may mediate p62‑dependent NF-κB activation. Moreover, we provide preliminary functional evidence that this signalling axis may promote CD8⁺ T-cell apoptosis and dysfunction in a tumour‑T cell co‑culture system.

P62/SQSTM1 is a multifunctional scaffold protein that plays a pivotal role in selective autophagy (13). It acts as both an autophagy substrate and a signalling hub. Under normal autophagy function, p62 binds to autophagosomes via its LC3-interacting region (LIR), ultimately being degraded by autophagolysosomes to maintain low intracellular levels. In the present study, MKN-45 cells showed lower basal p62 expression than GES-1 cells, which is attributed to cell-line-specific heterogeneity in GC. Notably, p62 elevation in clinical tumour tissues is often caused by autophagy impairment, inflammation, or hypoxia in the tumour microenvironment rather than inherent high expression in vitro (14). Our data shows that the pharmacological inhibition of autophagy by CQ further increases intracellular p62 and PD-L1 expression. Notably, short‑term treatment with low‑dose CQ (20 µM, 24 h) slightly promoted MKN45 cell proliferation, which may be attributed to p62 accumulation‑mediated NF-κB activation that supports adaptive survival and proliferation. This proliferative effect is context‑dependent: high concentrations or prolonged exposure to CQ usually induces lysosomal stress and cytotoxicity, whereas low‑dose shortterm treatment mainly blocks autophagic flux without overt cell damage (15). Thus, our observation is not contradictory but consistent with the dose‑ and timedependent effects of CQ reported in previous studies. The process of CQ-induced PD-L1 upregulation was significantly attenuated by p62 knockdown, supporting the potential essential role of p62 in this process. These findings are consistent with the concept that defective autophagy leads to p62 accumulation and the activation of oncogenic signalling in cancer cells (16,17). Specifically, in GC, Wang et al. reported that autophagy inhibition increased PD-L1 expression via p62/SQSTM1 and NF-κB (7), and our results in MKN-45 cells are consistent. We have shown that this link is confirmed in a defined cell model. Our comparison of MKN-45 and GES-1 suggests that elevated basal p62 may be a tumour intrinsic feature. This feature would predispose GC cells to upregulate PD-L1 when autophagy is impaired. Similar observations have been made in hepatocellular carcinoma and lung cancer, where p62 accumulation correlates with tumour progression and chemoresistance (18,19).

NF-κB is a key transcription factor in inflammatory and immune responses, and sustained activation of this factor is closely associated with the progression of various cancers and their ability to evade the immune system (20). We also show that p62 controls PD-L1 expression through NF-κB activation. Treatment with CQ increased p-IκBα and nuclear p65, indicating activation of the canonical NF-κB signalling pathway. However, the NF-κB inhibitor BAY 11-7082 effectively suppressed p65 activation and PD-L1 upregulation without affecting CQ-induced p62 accumulation. These findings suggest that NF-κB is downstream of p62 in this pathway. In GC, NFκB activation is frequently observed and associated with chronic inflammation and poor prognosis (21). While some studies have suggested that autophagy may influence NF-κB activity (22,23), the upstream processes were not fully explored. With regard to the correlation between NF-κB and PD-L1, earlier research has documented this association across various forms of cancer (24-27). Our data extend these observations by showing that p62 is not only required for CQ-induced NF-κB activation but also enhances the binding of p65 to the PD-L1 promoter, as demonstrated by ChIP-qPCR, thereby providing additional evidence for NF-κB-dependent transcriptional control of PD-L1 in GC cells. Notably, previous research has confirmed that inhibition of NFκB signaling is essential for oleanolic acid to downregulate PD-L1 expression via promoting DNA demethylation in GC cells, further supporting the critical role of NF-κB in regulating PD-L1 expression (28). This suggests that these events may occur during the early stages of GC development and continue to play a role throughout disease progression.

A key novel observation of this study is the identification of TRAF6 and RIP1 as p62‑associated adaptor proteins that may link p62 accumulation to NF-κB activation in MKN45 GC cells. Co-IP assays revealed that p62 interacts physically with TRAF6 and RIP1 under basal conditions and that these interactions are markedly strengthened upon autophagy inhibition. The association between p62 and TRAF6 was significantly weakened by domain mutation, highlighting the importance of this interaction. Immunofluorescence revealed enhanced co-localisation of p62 with TRAF6 and RIP1 after CQ treatment. To our knowledge, this study is the first to systematically demonstrate the p62–TRAF6/RIP1 complex as an upstream trigger for NF-κB-mediated PD-L1 expression in GC. Most previous investigations only focused on the linear correlation between p62 accumulation and downstream NF-κB activation (29), while ignoring the indispensable upstream adaptor molecules. Our findings fill this mechanistic gap and improve the integrity of the autophagy-p62-NF-κB-PD-L1 regulatory cascade in GC.

Another important functional contribution of this study is the direct evaluation of the p62/NF‑κB/PD‑L1 axis on CD8⁺ T‑cell responses. In an in vitro co-culture model with activated CD8⁺ T cells, CQ-treated MKN-45 cells markedly suppressed T-cell proliferation and promoted T-cell apoptosis, as well as reducing the secretion of IFN-γ and IL-10 while increasing TNF-α production. These alterations collectively drive CD8⁺ T-cell dysfunction and a pro-apoptotic phenotype. Consistent with previous findings, the binding of tumour cell PD-L1 to PD-1 on T cells impedes T-cell proliferation, disturbs cytokine secretion, and initiates T-cell apoptosis (30,31). Notably, BAY 11-7082 partially restored T-cell proliferation, reduced T-cell apoptosis, and regulated cytokine secretion when used in conjunction with CQ-treated tumour cells. Since BAY 11-7082 efficiently blocked CQ-induced PD-L1 upregulation in MKN-45 cells, our results demonstrate that hyperactivation of the p62/NF-κB/PD-L1 signalling cascade in GC cells may contribute to CD8⁺ T-cell dysfunction and tumour immune escape.

Unlike previous studies that mainly focused on molecular expression changes, we used a tumour-T cell co‑culture system to provide direct functional evidence supporting that the autophagy-p62-NF-κB-PD-L1 axis induces CD8⁺ T‑cell apoptosis and dysfunction in GC. We confirmed that autophagy inhibition-mediated p62/NF-κB/PD-L1 signalling directly induces CD8⁺ T cell apoptosis and functional exhaustion in GC, which provides novel and systematic immune functional evidence for this pathway and supplements the tumour immune escape mechanism in GC. Accumulating evidence from other tumour models has indicated that autophagy shapes anti-tumour immunity via regulating antigen presentation, type I interferon signalling and T-cell infiltration (32,33). Our results complement these studies by providing direct functional evidence that blocking autophagy and accumulating p62 in GC cells fosters a PD-L1-dependent immunosuppressive microenvironment at the T-cell proliferation and survival. The observed decrease in both IFN-γ and IL-10 in the CQ group suggests an impairment of T-cell effector function across the board, rather than a simple shift between Th1- and regulatory-type responses. Similar patterns of reduced cytokine production have been observed in exhausted CD8⁺T cells within the tumour microenvironment (34,35). The partial restoration of T‑cell function by NF-κB inhibition further supports the potential role of tumour‑intrinsic signalling in regulating T‑cell activity.

Naturally, this study has certain limitations that will need to be addressed in future work. Firstly, mechanistic analyses were focused on a single GC cell line (MKN-45), with GES-1 acting as a normal control. Notably, we have supplemented experimental verification in an additional HGC27 GC cell line, and confirmed that p62 expression was the most significant in MKN-45 cells, which further enhances the representativeness and credibility of the current cell model. While MKN-45 is a widely used model, validation in additional GC cell lines with different genetic profiles, as well as in vivo models, is necessary to confirm the general applicability of the p62-TRAF6/RIP1-NF-κB-PD-L1. Secondly, our work relied on co-culturing with T cells derived from healthy donors in vitro, which does not fully replicate the complexity of the tumour microenvironment, including stromal cells, regulatory T cells, and myeloid populations. Thirdly, the present study mainly evaluated PD-L1 expression at mRNA and total protein levels. Although these changes reliably reflect the upregulation of PD-L1 and are consistent with the observed immunosuppressive phenotypes, detection of cell surface PD-L1 by flow cytometry would provide more direct and functionally relevant evidence. This aspect will be further explored in future investigations. Fourthly, while ChIP-qPCR supports enhanced binding of p65 to the PD-L1 promoter, complementary assays such as PD-L1 promoter reporter analyses and mapping of specific κB motifs would strengthen the conclusion of direct transcriptional regulation further. Finally, our work focused on NF-κB as the major transcriptional mediator. Other transcription factors known to regulate PD-L1, such as STAT3, HIF-1α and AP-1, were not examined, but they may also interact with autophagy-related pathways. Future studies should therefore extend our observations to additional cellular and animal models and incorporate patient-derived tissues in order to assess clinical relevance. They should also explore combinatorial strategies targeting p62, NF-κB and PD-1/PD-L1 in preclinical immunotherapy settings. Additionally, it will be important to delineate how p62 interacts with other signalling networks, and whether specific p62 domains or post-translational modifications can be selectively targeted to modulate NF-κB and PD-L1 without impairing autophagy globally.

Conclusions

In summary, this study reveals the mechanistic link between the inhibition of autophagy and the upregulation of PD-L1 in GC. It identifies p62/SQSTM1-TRAF6/RIP1 complexes as upstream activators of NF-κB and demonstrates that activation of the p62/NF-κB/PD-L1 induces apoptosis and functional suppression of CD8⁺ T cells in vitro. These findings advance our understanding of how autophagy-related signalling contributes to tumour immune escape, suggesting that targeting p62-associated complexes and downstream NF-κB/PD-L1 signalling could enhance T cell-mediated immune responses and improve immunotherapeutic outcomes in this malignancy.

Acknowledgments

None.

Footnote

Reporting Checklist: The authors have completed the MDAR reporting checklist. Available at https://jgo.amegroups.com/article/view/10.21037/jgo-2026-1-0106/rc

Data Sharing Statement: Available at https://jgo.amegroups.com/article/view/10.21037/jgo-2026-1-0106/dss

Peer Review File: Available at https://jgo.amegroups.com/article/view/10.21037/jgo-2026-1-0106/prf

Funding: This work was supported by Clinical Research Project of Nanjing Medical University Changzhou Medical Center (No. CMCC202312), 2024 Changzhou Health Commission Science and Technology Project (No. ZD202415), and 2025 Changzhou 6th Batch Science and Technology Plan Projects (No. CJ20257003).

Conflicts of Interest: All authors have completed the ICMJE uniform disclosure form (available at https://jgo.amegroups.com/article/view/10.21037/jgo-2026-1-0106/coif). The authors have no conflicts of interest to declare.

Ethical Statement: The authors are accountable for all aspects of the work in ensuring that questions related to the accuracy or integrity of any part of the work are appropriately investigated and resolved.

Open Access Statement: This is an Open Access article distributed in accordance with the Creative Commons Attribution-NonCommercial-NoDerivs 4.0 International License (CC BY-NC-ND 4.0), which permits the non-commercial replication and distribution of the article with the strict proviso that no changes or edits are made and the original work is properly cited (including links to both the formal publication through the relevant DOI and the license). See: https://creativecommons.org/licenses/by-nc-nd/4.0/.

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