Abstract
Background
The PSMD11 has been shown to be associated with the malignant progression and clinical outcomes of various carcinomas. However, the molecular mechanism of PSMD11 in non-small cell lung cancer (NSCLC) remains unknown. In this study, we investigated the molecular mechanism of PSMD11 effect on the immune escape in NSCLC.
Methods
Co-culture models of NSCLC cells and T cells were constructed. Colony formation assays and flow cytometry were used to detect the proliferation and apoptosis of NSCLC cells in the co-culture system after the knock down of PSMD11. Western blotting was used to detect the expression of programmed death-ligand 1 (PD-L1) after the knock down of PSMD11. Co-immunoprecipitation (co-IP) assays were used to observe the interaction between PD-L1 and PSMD11 and USP14 in the A549 and H1299 cells. The in vivo effects of PSMD11 were analyzed using a xenograft tumor model.
Results
In the NSCLC cell/T cell co-culture models, the knockdown of PSMD11 significantly upregulated T cell-mediated apoptosis and enhanced the killing effect of T cells in the A549 and H1299 cells, and also significantly upregulated the expression of killer T cell markers Granzyme and perforin in the T cells. We also found that the knockdown of PSMD11 inhibited the expression of PD-L1. The mass spectrometry (MS) and co-IP results showed that PSMD11 interacted with PD-L1 and the deubiquitinating enzyme USP14. Overexpression of USP14 leads to an increase in the protein level of PD-L1, while knocking down PSMD11 can reverse this elevated state. The overexpression of PD-L1 and USP14 in the NSCLC cells reversed the effects of PSM11 on immune escape. In vivo, the knockdown of PSMD11 promoted the anti-tumor effect of anti-PD-1 therapy.
Conclusions
This study showed that PSMD11 recruits USP14 to modulate the deubiquitinating degradation of PD-L1 to promote immune escape in NSCLC.
Keywords: PSMD11, USP14, programmed death-ligand 1 (PD-L1), immune escape, non-small cell lung cancer (NSCLC)
Highlight box.
Key findings
• This study showed that PSMD11 recruits USP14 to modulate the deubiquitinating degradation of programmed death-ligand 1 (PD-L1), thereby promoting immune escape in non-small cell lung cancer (NSCLC).
What is known, and what is new?
• Previous research has shown that PSMD11 is related to the malignant development and clinical prognosis of various cancers.
• The present study revealed that PSMD11 recruits USP14 into the cell, thereby regulating the de-ubiquitination degradation process of PD-L1, and subsequently promoting immune escape in NSCLC.
What is the implication, and what should change now?
• Further studies on the interaction between PSMD11 and USP14, as well as the development of new small molecule inhibitors or gene therapies, may lead to more personalized and effective immunotherapy options for NSCLC patients.
Introduction
Lung cancer is the most common cancer, and the leading cause of cancer-related morbidity and mortality worldwide (1). Non-small cell lung cancer (NSCLC) is the most common type of lung cancer, and accounts for about 85% of all lung cancers (2,3). Despite recent advancements and the practical implementation of novel therapeutic approaches (e.g., screening, less intrusive methods for diagnosis and treatment, improvements in radiation therapy, novel molecularly targeted medications, and immune checkpoint inhibitors), pertinent epidemiological data show that NSCLC patients have a poor prognosis due to late diagnosis and resistance (whether innate or acquired) to modern anti-cancer therapy (4,5). Specifically, although immune checkpoint inhibitors have revolutionized the treatment of various malignant tumors, their effectiveness is limited due to the complexity of the immune system and the diversity of tumor immune evasion. As a result, the population that can benefit from these treatments is relatively small. The significant heterogeneity of both the genetic composition within the tumor and the microenvironment between tumors that dictates many of the cellular and molecular features of the illness is a primary treatment problem for NSCLC. For instance, some NSCLC patients develop resistance to PD-1 inhibitors due to mutations in the β2-microglobulin gene of tumor cells, which leads to the absence of MHC-I molecule expression and prevents T cells from recognizing tumor antigens; while for another group of patients, it is because of the abundant enrichment of myeloid-derived suppressor cells (MDSCs) in the tumor microenvironment, or the upregulation of alternative immune checkpoints (such as LAG-3, TIM-3), that inhibits the function of T cells.
Eukaryotic cells are the basic structural and functional units that constitute animals, protozoa, and other eukaryotic organisms. Their complex and highly efficient life activities not only support the independent survival of single-celled eukaryotic organisms, but also promote cell differentiation, tissue formation, and organ development in multicellular organisms. Tumor cells, as eukaryotic cells, have complex structures and highly specialized functions. Their normal operation relies on the precise regulation of tens of thousands of proteins. And the proteasome is the core molecular device that eukaryotic cells have evolved to meet this requirement. The ubiquitin-proteasome system (UPS) is an indispensable protein turnover and degradation pathway that is tightly regulated in various intracellular processes and proteins that dominate human antigen processing, signal transduction, and cell-cycle regulation in eukaryotic cells (6). The UPS consists of specific enzymes that modify protein substrates. Ubiquitin and proteasomes are responsible for the proteolysis of the ubiquitin-tagged substrates that take part in different physiological processes, including apoptosis, angiogenesis, as well as antigen presentation and DNA damage control. Currently, many studies have shown that the malfunction and altered expression of the UPS are significantly associated with malignant progression and tumor immune microenvironment heterogeneity in various carcinomas (7,8).
The 26S proteasome is composed of a cylindrical 20S proteolytic core particle and one or two regulating 19S regulatory particles, and is responsible for the degradation of intracellular proteins that maintain proteostasis (9). The 20S core is constructed from inner α-rings and outer β-rings, which are both divided into seven structurally similar subunits, namely the proteasome 20S subunits α (PSMA1-7) and β (PSMB1-7). The 19S cap complexes are composed of a base and a lid subcomplex, further categorized into ATPase subunits (PSMC1–6) and non-ATPase subunits (PSMD1–14). PSMD11 protein, also known as RPN-6 in Caenorhabditis elegans, has been shown to be an important factor in cancer cell survival (10). The PSMD11 protein has been found to be associated with proliferation, apoptosis, and cell-cycle progression in cancer cells, and its knockdown has been shown to inhibit proliferation, migration, invasion, and tumor growth in NSCLC cells (11). Such evidence suggests that PSMD11 may be a multi-functional protein and could serve as a novel therapeutic target for NSCLC. However, further research needs to be conducted to determine whether PSMD11 can be targeted to treat NSCLC. Additionally, the effect of PSMD11 on immune escape in NSCLC remains unknown.
In this study, we investigated the effect of PSMD11 expression on the immune response in NSCLC cell/T cell co-culture models. In this co-culture model, we used NSCLC cell lines and T cells isolated from human peripheral blood (with a T cell:cancer cell ratio of 10:1). We also detected PDL-1 and USP14 as the key downstream molecules of PSMD11 by mass spectrometry (MS) and co-immunoprecipitation (co-IP). The experiment results revealed an interaction between PD-L1 and PSMD11 and the deubiquitinating enzyme USP14. Furthermore, we found that compared to the Control group where PSMD11 was not knocked down, knocking down PSMD11 would enhance the anti-tumor effect of anti-PD-1 therapy in vivo. Further, we found that the knockdown of PSMD11 promotes the anti-tumor effects of anti-PD-1 therapy in vivo. To summarize, this study revealed that PSMD11 plays a role in NSCLC progression, such that PD-L1 expression is upregulated by PSMD11 and USP14, resulting in tumor immune escape. We present this article in accordance with the ARRIVE and MDAR reporting checklists (available at https://jtd.amegroups.com/article/view/10.21037/jtd-2025-1672/rc).
Methods
Cell treatment
Human NSCLC cell lines A549 and H1299, and Lewis cells from a mouse lung cancer cell line all originated from the Chinese Academy of Sciences. They were cultured and expanded in Dulbecco’s modified Eagle’s medium (DMEM, Hyclone, Visalia, CA, USA), supplemented with 10% fetal bovine serum (FBS, Hyclone) in a 5% carbon dioxide (CO2) humidified incubator (Thermo, Waltham, MA, USA) at 37 ℃.
Cell transfection
The short-interfering RNA (siRNA) for BANP (siBANP) and PMSD11 (siPSMD11), and the negative control (siNC) were purchased from Tsingke (Beijing, China). The PD-L1 overexpression vector (pcDNA3.1-PD-L1), USP14 overexpression vector (pcDNA3.1-USP14) purchased from Miaoling (Wuhan, China). The cancer cells were seeded into 6-well plates until they reached 50% confluence in normal culture medium. According to the instructions for use, the transfection mixture for each well comprised 20 µL of lipofectamine 2000 (Invitrogen, Carlsbad, CA, USA), 50 nM of siRNAs, and 980 µL of Opti-MEM (Thermo). After transfection for 8 hours, the medium was replaced with complete culture medium and incubated for another 40 hours. The transfection efficacy was measured by quantitative real-time polymerase chain reaction (qRT-PCR) and western blot assays.
T cell co-culture
Human peripheral blood mononuclear cells (Hycells Biotech, Shanghai, China) were purified from peripheral blood samples using the Ficoll extraction method, and the T cells were then isolated using a Dynabeads™ Untouched™ human T cell isolation kit (#11344D, Thermo) in accordance with the manufacturer’s instructions. The T cells were cultured in Roswell Park Memorial Institute-1640 medium containing 10% FBS at 37 ℃ with 5% CO2 in an incubator. For the co-culture, the T cells were placed in a 6-well Transwell chamber (0.40-µm pore size, Costar), and the cancer cells were seeded in the bottom chamber (at a T cell:cancer cell ratio of 10:1). After co-culture for 3 days, the cells were collected for subsequent experiments.
Colony formation assays
These cells were subjected to trypsin digestion and suspended as single cells in culture DMEM. A total of 1,000 cells were seeded into each well of a 6-well plate and incubated at 37 ℃ in an incubator. After incubation for 10 days, the colonies were washed with phosphate-buffered saline (PBS) and stained with crystal violet (C0120, Beyotime). The stained colonies were washed with PBS and water, and then underwent natural withering. Images were captured with a digital camera (Leica, Germany).
Cell apoptosis detection
Cell apoptosis was analyzed using the annexin V-FITC apoptosis detection kit (C1062S, Beyotime). In brief, the cells were collected and suspended in annexin V-FITC binding buffer containing annexin V-FITC and PI, and incubated in the dark at room temperature for 20 minutes. The samples were then analyzed by BD-FACS flow cytometry (BD Bioscience, Franklin, NJ, USA).
RNA isolation and qRT-PCR
The tumor tissues and cells were lysed with TRIzol reagent (Invitrogen) to isolate the total RNA in accordance with the manufacturer’s instructions. For the synthesis of complementary DNA (cDNA), 1 µg of total RNA was reverse-transcribed with the Thermo Scientific RevertAid RT kit (#K1691, Thermo). RNase-free distilled water was used to prepare a 10-fold dilution of the resulting cDNA. QRT-PCR was then conducted using the SuperScript™ III Platinum™ kit (#11732088, Thermo) in accordance with the manufacturer’s instructions. The relative expression levels of RNA were determined using the 2-∆∆Ct method and normalized to GAPDH. The primers used were synthesized by RiboBio (Guangzhou, China).
The primer sequences were as follows:
PD-L1: forward: 5'-TGGCATTTGCTGAACGCATTT-3', reverse: 5'-TGCAGCCAGGTCTAATTGTTTT-3'; PSMD11: forward: 5'-TCGCCTGGTCCGATCTCTT-3', reverse: 5'-ATGCACTCTAAACACAGCTCG-3'; CD276: forward: 5'-CTGGCTTTCGTGTGCTGGAGAA-3', reverse: 5'-GCTGTCAGAGTGTTTCAGAGGC-3'; GAPDH: forward: 5'-GGAGCGAGATCCCTCCAAAAT-3', reverse: 5'-GGCTGTTGTCATACTTCTCATGG-3'.
Western blot assays
The total proteins were extracted from the tumor tissues and cells using RIPA lysis buffer. The reason for choosing RIPA buffer instead of NP-40 buffer is that our target protein (PSMD11) has some nuclear localization characteristics. The stronger solubilization ability of RIPA buffer can ensure the simultaneous extraction of both the cytoplasm and the nuclear part, while the NP-40 buffer mainly extracts cytoplasmic proteins. The protein concentrations were subsequently measured using the bicinchoninic acid protein assay kit (Thermo). Carry out at least three parallel experiments. An equal number of total proteins were loaded and separated by 10% sodium dodecyl sulfate-polyacrylamide gel electrophoresis and then transferred to polyvinylidene difluoride membranes (Millipore, Bedford, MA, USA). The membranes were then blocked with 5% non-fat milk at room temperature for 2 hours and probed with anti-PSMD11 (1:2,000, ab221645, Abcam), Granzyme B (ab255598, Abcam), perforin (ab256453, Abcam), anti-PD-L, anti-USP14, and anti-GAPDH (1:2,000, ab9485, Abcam) antibodies overnight at 4 ℃. The next day, the protein bands were treated with the following horseradish peroxidase conjugated secondary antibodies at room temperature for 1 hour: goat-anti-mouse (ab6789, Abcam) and goat-anti-rabbit (ab6721, Abcam). Afterward, the protein bands were detected using the SuperSignal West Pico PLUS reagent (#34577, Thermo).
Co-IP assays
The co-IP assays were conducted using the Dynabeads Antibody Coupling Kit (Thermo, Waltham, MA, USA) in accordance with the manufacturer’s instructions. Anti-immunoglobulin G (anti-IgG), anti-PSMD11, and anti-USP14 antibodies were allowed to bind to the beads at 37 ℃ on a roller for a period of 6 hours. Subsequently, the cancer cells were subjected to transfection with specific siRNAs or expression vectors, after which they were exposed to MG132 (5 µM) for 6 hours. The cells were then lysed using RIPA buffer, and the lysates were incubated with the antibody-coated beads. Following a series of washes, the beads were retrieved, and the associated protein complexes were eluted and subsequently analyzed by western blot.
Immunohistology experiment
The fixed tissues were dehydrated and embedded in paraffin, and then cut into 5-µm thick slices. The tissue samples were immersed in citrate sodium buffer for 10 minutes for the antigen retrieval and 3% H2O2 for the endogenous peroxidase inactivation. The tissues were then blocked with goat serum for 30 minutes at room temperature and probed with anti-PD-L1 antibody (ab205921, Abcam) at 4 ℃ overnight. The next day, the samples were reacted with anti-rabbit IgG (biotin) secondary antibody (ab97049, Abcam) at room temperature for 1 hour. Images were captured with a microscope (Leica).
Mouse model and treatments
Male C57BL/6J mice, aged 4–6 weeks old, were bought from Vital River Laboratory (Beijing, China) and acclimated in a specific pathogen-free animal facility for one week. Specifically, a randomization sequence was generated using a random number table, and based on this sequence, the mice were divided into four groups with 5 mice in each group. A total of 5×106 Lewis cells were seeded in the right-side fat pad of each mouse. The width and length of the tumors were measured and recorded every three days. When the tumor size reached 100 mm3, treatment with siPSMD11 (200 nmol/kg body weight, every 3 days) and PD-1 monoclonal antibody (5 mg/kg body weight, every 5 days) were administrated via intratumor injection and intraperitoneal injection, respectively. After inoculation for 20 days, the mice were sacrificed, and the tumors were collected for subsequent experiments. The order of mouse treatment and tumor volume measurement was determined using a random number table. Meanwhile, the cage positions of the control group and treatment group were swapped every 3 days to reduce the confounding effect of location-related environmental factors. During the group allocation stage, only the statisticians were aware of the group assignments. In the experiment implementation stage (animal dosing), dedicated personnel performed the operations according to codes and were unaware of the group assignments. During the outcome assessment stage (tumor measurement) and data analysis stage, all relevant personnel remained blinded (i.e., unaware of the group assignments) until the data were locked, after which the blinding was unblinded. Experiments were performed under a project license (No. KY2019SL058-02) granted by the Institutional Animal Care and Use Committee of Ningbo Medical Center LihuiLi Hospital, in compliance with the “Guidelines for the Care and Use of Laboratory Animals” formulated by the Laboratory Animal Research Institute for the care and use of animals. A protocol was prepared before the study without registration
Flow cytometry of tumor-infiltrating T cells
The Xenograft tumors were excised from the mice and diced into small pieces, and then ground in cell suspension with PBS. To match the size of the target cells, these cells were filtered through a 70-µm filter, resulting in a single-cell suspension, fixed and penetrated with eBioscience Intracellular fixation rupture buffer (#88-8824-00, Thermo), and incubated with APC anti-human CD3 antibody (100236, BioLegend), FITC anti-mouse CD8b.2 antibody (140404, BioLegend), PE anti-human/mouse Granzyme B (372208, BioLegend), and PE anti-mouse perforin antibody (154306, BioLegend) for the cytotoxic T cells. After staining for 30 minutes, the cells were centrifuged, resuspended in PBS, and analyzed with a BD-FACS flow cytometer (BD Bioscience, Franklin, NJ, USA). The data were analyzed using FlowJo software.
Statistical analysis
The data in this study are presented as the mean ± standard deviation (SD), and they include the results of three independent repeated experiments. The statistical analysis was conducted using the SPSS 19.0 software. Intergroup comparisons were conducted using the Student’s t-test, while multiple comparisons were analyzed by one-way analysis of variance followed by Tukey’s post-hoc test. A P value less than 0.05 was considered statistically significant.
Results
PSMD11 plays a role in promoting immune suppression in NSCLC
To investigate the effect of PSMD11 on immune escape in NSCLC, we analyzed the effect of PSMD11 on the killing of lung cancer cells by T cells in the NSCLC cell/T cell co-culture models. Specifically, we used siRNAs to knock down the expression of PSMD11 in the A549 and H1299 cells; PSMD11 siRNA-3 was selected for use in the subsequent studies because it had the highest knockdown efficiency (Figure 1A). The apoptosis analysis indicated that the knockdown of PSMD11 led to apoptosis in A549 and H1299 cells. Moreover, in the co-culture model of NSCLC cells and T cells, the knockdown of PSMD11 in A549 and H1299 cells enhanced the apoptosis mediated by T cells. (Figure 1B,1C). Similarly, the cloning formation experiments showed that PSMD11 knockdown inhibited the proliferative ability of the A549 and H1299 cells, while PSMD11 knockdown significantly enhanced the killing effect of the T cells in the A549 and H1299 cells in the NSCLC cell/T cell co-culture models. The proliferation of the A549 and H1299 cells was also reduced (Figure 1D,1E). Importantly, in the NSCLC cell/T cell co-culture models, PSMD11 knockdown significantly upregulated the expression of killer T cell markers Granzyme and perforin in the T cells (Figure 1F).
Figure 1.
PSMD11 expression facilitates immune escape in NSCLC. (A) NSCLC cells (A549 and H1299) were transfected with siPSMD11, and PSMD11 protein expression was detected by western blot. (B,C) The apoptotic A549 cells and H1299 cells were analyzed by flow cytometry. (D,E) The proliferation of the A549 and H1299 cells was analyzed by the colony formation assay (stained with crystalline violet). (F) The expression of killer T cell markers Granzyme and perforin in T cells were detected by western blot. ***, P<0.001. FITC, fluorescein isothiocyanate; NSCLC, non-small cell lung cancer; PI, propidium iodide; V-FITC, fluorescein 5-isothiocyanate.
Knockdown of PSMD11 inhibits the expression of PD-L1
To explore the molecular mechanism by which PSMD11 expression promotes immune escape in NSCLC, we analyzed the effect of PSMD11 on checkpoint expression in the NSCLC cell/T cell co-culture models. In the NSCLC/T cell co-culture models, we found that knocking down the expression of PSMD11 inhibited the expression of PD-L1 as analyzed by western blot, and the level of PD-L1 on the cell membrane as analyzed by flow cytometry (Figure 2A,2B). These results suggest that the knockdown of PSMD11 promotes the killing effect of T cells in NSCLC cells and the expression of PD-L1 in lung cancer cells in vitro.
Figure 2.
Knockdown of PSMD11 inhibits the expression of PD-L1. (A) The expression of PD-L1 in the A549 cells and H1299 cells was analyzed by western blot. (B) The levels of PD-L1 on the cell membrane of the A549 cells and H1299 cells were analyzed by flow cytometry. ***, P<0.001. PD-L1, programmed death-ligand 1.
PSMD11 modulates the stability of PD-L1 by introducing USP14
To determine the regulatory mechanism of PSMD11 in PD-L1 expression, we performed a co-IP experiment and observed the interaction between PD-L1 and PSMD11 (Figure 3A) and the deubiquitinating enzyme USP14 (Figure 3B) in the A549 and H1299 cells. The knockdown of PSMD11 with siRNA led to the decreased expression of PD-L1 in whole cell lysates (Figure 3C). Notably, the knockdown of PSMD11 did not affect the expression of USP14 but did reduce the binding of PD-L1 to USP14 (Figure 3C). Subsequently, we analyzed the role of USP14 in regulating PD-L1 expression. Overexpression of USP14 leads to an increase in the protein level of PD-L1, while knocking down PSMD11 can reverse this elevated state (Figure 3D). Additionally, we used MG132 to inhibit proteasome-mediated protein degradation, and measured the PS-L1 protein stability under the alteration of USP14 and PSMD11. As Figure 3E shows, the overexpression of USP14 suppressed the ubiquitination and degradation of PD-L1. These results indicated that PSMD11 modulated the degradation of PD-L1 by recruiting USP14.
Figure 3.
PSMD11 modulates the stability of PD-L1 by introducing USP14. (A,B) A co-IP experiment was conducted to examine the interaction between PSMD11 and PD-L1 and USP14 in the lung cancer cells. (C) A co-IP experiment was conducted to measure the binding of USP14 to PD-L1 and USP14 in the lung cancer cells that had been transfected with NC or siPSMD11. (D) The A549 and H1299 cells were treated with USP14 overexpression and siPSMD11, and PD-L1 expression was assessed by western blot assay. (E) The A549 and H1299 cells were treated with MG132, and the ubiquitination of PD-L1 was then measured. co-IP, co-immunoprecipitation; HA, hemagglutination; IB, immunoblotting; NC, negative control; PD-L1, programmed death-ligand 1; WCL, whole cell lysates.
PSMD11 regulates lung cancer cell immune evasion via the USP14/PD-L1 axis
Next, we established a cancer cell-T cell co-culture models and measured the cell killing effects of the T cells on the lung cancer cells. The colony formation and flow cytometry results showed that the knockdown of PSMD11 led to decreased proliferation and increased apoptosis in the A549 and H1299 cells, suggesting enhanced sensitivity of cancer cells to T cells, while the overexpression of PD-L1 and USP14 in the cancer cells reversed these effects (Figure 4A,4B). Further, the co-culture with the cancer cells that silenced PSMD11 significantly elevated the production of granzyme B and perforin by the T cells, compared with the control cells, which was reversed by the overexpression of PD-L1 and USP14 (Figure 4C). Notably, the knockdown of PSMD11 reduced the expression of PD-L1 and CD276 (another immune checkpoint protein) in the lung cancer cells, and the overexpression of USP14 recovered their expression (Figure 4D).
Figure 4.
PSMD11 regulates lung cancer cell immune evasion via the USP14/PD-L1 axis. Co-culture models of T cells with A549 and H1299 cells were established. (A) The proliferation of cancer cells was measured by colony formation assay, performed crystal violet staining on the cell clones to display the number of cell clones. (B) The apoptosis of cancer cells was measured by flow cytometry. (C) The production of granzyme B and perforin by T cells was detected by western blot. (D) The protein levels of PD-L1 and CD276 in cancer cells were detected by western blot. **, P<0.01 vs. NC group; ##, P<0.01 vs. siPSMD11 group. NC, negative control; PD-L1, programmed death-ligand 1.
Knockdown of PSMD11 promotes the anti-tumor effects of anti-PD-1 therapy
We next analyzed the in vivo effects of PSMD11 using a xenograft tumor model. We observed that treatment with either siPSMD11 or anti-PD-1 alone notably suppressed growth and tumor volume in vivo, compared with the untreated group (Figure 5A). The combination of siPSMD11 and anti-PD-1 exerted a synergistic anti-tumor effect (Figure 5A), and also suppressed the protein levels of PD-1 and PSMD11 (Figure 5B). Additionally, the mono-treatment with siPSMD11 or anti-PD-1 elevated the portion of CD8+/granzyme B+ T cells and CD8+/perforin T cells in the tumor tissues, and the combined therapy significantly enhanced these effects (Figure 5C).
Figure 5.
The knockdown of PSMD11 promotes the anti-tumor effects of anti-PD-1 therapy. A xenograft tumor model was established and treated with siPSMD11 or/and anti-PD-1 antibody. (A) Tumor growth and tumor volume were measured. (B) Protein expression of PSMD11 and PD-L1 in tumor tissues was detected by western blot. (C) The portion of CD8+/granzyme B+ T cells and CD8+/perforin T cells in tumor tissues was measured by flow cytometry. **, P<0.01 vs. NC group; #, P<0.05 vs. anti-PD-1 group; ##, P<0.01 vs. anti-PD-1 group. NC, negative control; PD-1, programmed death-1; PE, phycoerythrin.
Discussion
NSCLC is the most common subtype of lung cancer, and is characterized by high heterogeneity and complex molecular mechanisms (12,13). Common clinical treatment methods for NSCLC include surgery, chemotherapy, targeted therapy, and immunotherapy (14). However, the therapeutic efficacy of NSCLC is often limited by the high degree of tumor heterogeneity and complex molecular mechanisms. The heterogeneity and complex molecular mechanisms of NSCLC are the core reasons for the difficulty in clinical treatment and the significant differences in prognosis. In the tumor cells of the same patient, different subclones may carry different gene mutations (such as EGFR sensitive mutations and T790M drug-resistant mutations coexisting). When using targeted drugs, the sensitive subclones are inhibited, while the drug-resistant subclones proliferate rapidly, ultimately leading to treatment failure. At the same time, the heterogeneity among tumors causes significant differences in response to the same treatment plan among different patients. Some NSCLC patients respond persistently to immune checkpoint inhibitors (PD-1 antibodies), while others never respond due to the enrichment of immunosuppressive cells (such as Treg cells) in the tumor microenvironment. Moreover, there are multiple core oncogenic pathways in NSCLC (such as PI3K-AKT-mTOR and RAS-RAF-MEK-ERK pathways), and these pathways do not act independently but jointly regulate tumor cell proliferation, apoptosis, and invasion through “cross-talk”. In recent years, immunotherapy, especially the application of programmed cell death receptor 1 and its ligand PD-L1 (PD-1/PD-L1) inhibitors has made significant progress in the treatment of NSCLC (15). However, immune escape remains an important mechanism by which tumors evade immune surveillance, leading to limitations in immunotherapy.
Programmed death-ligand 1 (PD-L1) is a glycoprotein located on the cell membrane, which is widely expressed in many types of tumor cells. PD-L1 inhibits the proliferation, differentiation, and cytotoxic activity of T cells by binding to the PD-1 receptor on the surface of T cells, thereby weakening the ability of the immune system to attack tumor cells (16). This action helps tumor cells to evade immune surveillance, which eventually leads to the phenomenon of tumor immune escape.
The proteasome system is the main pathway for protein degradation in cells and is essential for maintaining cell homeostasis (17). As a regulatory subunit of the 20S proteasome, PSMD11 is involved in the process of protein recognition and degradation (18). Recent studies have shown that PSMD11 is abnormally expressed in a variety of cancers (10,11,19). The high expression of PSMD11 is associated with tumor proliferation, invasion, and chemotherapy resistance. Therefore, PSMD11 may affect tumor progression and the treatment response by regulating protein degradation. Specifically, in liver cancer, PSMD11 can promote the degradation of tumor suppressor proteins (such as p21 and p27) by the proteasome, reducing the accumulation of these proteins and thereby relieving their inhibition on the cell cycle, facilitating the transition of tumor cells from the G1 phase to the S phase, and accelerating cell proliferation. In pancreatic cancer research, high expression of PSMD11 can enhance the resistance of tumor cells to gemcitabine. The mechanism is that PSMD11 promotes the degradation of drug-induced apoptosis proteins (such as caspase-3 precursor) by the proteasome, inhibiting the activation of the apoptotic pathway.
In NSCLC, the role of PSMD11 has not been fully studied, but its key regulatory role in the proteasome system makes it a potential candidate for investigation (11). In the present study, we found that PSMD11 plays a role in promoting immunosuppression in NSCLC. Further, we found that PSMD11 may synergistically interact with the deubiquitinating enzyme USP14 to regulate the degradation of key proteins, thereby affecting the immune escape mechanism of tumors.
USP14 is a key deubiquitinating enzyme in the UPS that removes ubiquitin chains from proteins, thereby preventing protein degradation (20,21). USP14 is abnormally expressed in a variety of tumors, and studies have shown that its overexpression is closely related to tumor cell proliferation, migration, and drug resistance (22,23). In our study, USP14 affected immune escape by regulating the ubiquitination and degradation of the immune checkpoint protein PD-L1. PD-L1 is an important immune checkpoint protein that inhibits the activity of T cells by binding to PD-1, thereby evading immune surveillance. By removing the ubiquitin chain from PD-L1, USP14 prevents its degradation by the proteasome, leading to the accumulation of PD-L1 on the cell surface, which contributes to the occurrence of tumor immune escape.
PSMD11 is a key regulatory factor of the USP14/PD-L1 axis that we discovered in our mechanism research. However, the clinical application of PSMD11 depends on the technical feasibility of its detection and its compatibility with existing clinical laboratory facilities. Firstly, we systematically evaluated the mainstream detection techniques for PSMD11 and their applicability in the clinical environment. Immunohistochemistry (IHC) may become the most suitable method for clinical application, mainly because it is compatible with formalin-fixed paraffin-embedded, which is the standard preservation form for clinical specimens of NSCLC. Additionally, almost all NSCLC diagnosis centers are equipped with IHC platforms, requiring no additional expensive equipment. In contrast, flow cytometry (FCM) requires fresh single-cell samples (such as tumor-infiltrating lymphocytes), which are difficult to obtain routinely in clinical practice; while Western Blot requires a large amount of tissue samples (>10 milligrams), which is not suitable for small needle biopsy and thus is not suitable for routine clinical application. However, there are also practical problems in clinical PSMD11 detection. Firstly, there is a limited sample size issue. Secondly, the standardization among different laboratories varies. To solve these practical problems, future efforts should optimize the IHC protocol, reduce the tissue consumption of the sections, while maintaining the signal intensity. Develop detailed IHC operation procedures to improve the consistency between laboratories. Further enhance the clinical value of PSMD11.
The synergistic effect of PSMD11 and USP14 may play an important role in the degradation of PD-L1. First, as a regulatory subunit of the proteasome, PSMD11 can recognize and direct PD-L1 for degradation. However, USP14, through its function as a deubiquitinating enzyme, removes the ubiquitin chain from PD-L1, thereby protecting PD-L1 from degradation. This mechanism increases the expression of PD-L1 on the surface of tumor cells, and then inhibits the killing function of T cells by binding to PD-1 on T cells, ultimately leading to tumor immune escape.
Conclusions
This study showed that PSMD11 recruits USP14 to modulate the deubiquitinating degradation of PD-L1 to promote immune escape in NSCLC. PSMD11/USP14 inhibits the ubiquitination degradation of PD-L1, and stabilizes the protein expression level of PD-L1, thus affecting the proliferation of NSCLC. Further research should be conducted into the interaction between PSMD11 and USP14, and the development of novel small molecule inhibitors or gene therapies may provide more personalized and effective immunotherapy regimens for NSCLC patients.
Supplementary
The article’s supplementary files as
Acknowledgments
None.
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. Experiments were performed under a project license (No. KY2019SL058-02) granted by the Institutional Animal Care and Use Committee of Ningbo Medical Center Lihuili Hospital, in compliance with the “Guidelines for the Care and Use of Laboratory Animals” formulated by the Laboratory Animal Research Institute for the care and use of animals.
Reporting Checklist: The authors have completed the ARRIVE and MDAR reporting checklists. Available at https://jtd.amegroups.com/article/view/10.21037/jtd-2025-1672/rc
Funding: This study was supported by funding from the Ningbo Clinical Research Center for Thoracic and Breast Neoplasms (No. 2021L002), the Ningbo Natural Science Foundation (No. 2022J256), and the NINGBO Medical and Health Leading Academic Discipline Project (No. 2022-F02).
Conflicts of Interest: All authors have completed the ICMJE uniform disclosure form (available at https://jtd.amegroups.com/article/view/10.21037/jtd-2025-1672/coif). The authors have no conflicts of interest to declare.
(English Language Editor: L. Huleatt)
Data Sharing Statement
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