MEN1 deficiency stabilizes PD‐L1 and promotes tumor immune evasion of lung cancer

Abstract

Multiple Endocrine Neoplasia 1 gene (MEN1), which is known to be a tumor suppressor gene in lung tissues, encodes a 610 amino acid protein menin. Previous research has proven that MEN1 deficiency promotes the malignant progression of lung cancer. However, the biological role of this gene in the immune microenvironment of lung cancer remains unclear. In this study, we found that programmed cell death‐ligand 1 (PD‐L1) is upregulated in lung‐specific Kras ^G12D mutation‐induced lung adenocarcinoma in mice, after Men1 deficiency. Simultaneously, CD8⁺ and CD3⁺ T cells are depleted, and their cytotoxic effects are suppressed. In vitro, PD‐L1 is inhibited by the overexpression of menin. Mechanistically, we found that MEN1 inactivation promotes the deubiquitinating activity of COP9 signalosome subunit 5 (CSN5) and subsequently increases the level of PD‐L1.

MEN1 deletion accelerates the progression of Kras‐induced lung adenocarcinoma, deregulates programmed cell death‐ligand 1 (PD‐L1), and modifies the lung tumor microenvironment in mice. These changes ultimately result in the early depletion of CD8⁺, CD3⁺ and CD4⁺ T cells. Mechanistically, menin promotes PD‐L1 destabilization and degradation by inhibiting expression of deubiquitinating enzyme COP9 signalosome subunit 5 (CSN5).

Abbreviations

AT II

alveolar type 2

CHX

cycloheximide

Co‐IP

coimmunoprecipitation

CSN5

COP9 signalosome subunit 5

DUB

deubiquitinating enzyme

GO

Gene Ontology

GSEA

Gene Set Enrichment Analysis

ICI

immune checkpoint inhibitor

IFN‐γ

γ‐interferon

IHC

immunohistochemistry

KM

Kaplan–Meier

KMS

LSL‐Kras ^G12D/+, Men1 ^f/f, Sftpc‐Cre

KS

LSL‐Kras ^G12D/+ Sftpc‐Cre

LUAD

lung adenocarcinoma

NK

natural killer

NSCLC

non‐small‐cell lung cancer

PD‐1

programmed cell death‐1

PD‐L1

programmed cell death‐ligand 1

PTM

posttranslational modification

qPCR

quantitative PCR

RNA‐seq

RNA sequencing

TAM

tamoxifen

TCGA

The Cancer Genome Atlas

TME

tumor microenvironment

TNF‐α

tumor necrosis factor‐α

Ub

ubiquitin

1. INTRODUCTION

Immunotherapies that target the PD‐1/PD‐L1 axis can reactivate T cells and restore the cytotoxic effects of T cells to kill tumor cells; these therapies have shown remarkable anti‐lung tumor effects. ¹ However, in clinical practice, approximately 50%–75% of NSCLC patients do not clearly benefit from this treatment approach. ² Thus, further understanding the mechanisms that regulate PD‐L1 expression in NSCLC is critical. The levels of PD‐L1 expression in tumor cells are regulated by various factors. Among PTMs, ubiquitination and deubiquitination are the most essential modifications, and they have been validated to play pivotal roles in regulating PD‐1/PD‐L1 protein stability and T cell antitumor functions. ³ , ⁴ , ⁵ CSN5 is a deubiquitinating enzyme that can mediate the removal of Ub from Ub‐conjugated substrate proteins. ⁶ , ⁷ Moreover, previous studies have reported that CSN5 reduces PD‐L1 ubiquitination and stabilizes PD‐L1, which further results in immune evasion. ⁸ , ⁹

Emerging studies have shown that MEN1 is an important tumor suppressor gene in lung tissues, and is extensively involved in gene expression, ¹⁰ protein stability, ¹¹ cellular proliferation, ¹² , ¹³ differentiation, ¹⁴ and migration. ¹⁵ Importantly, MEN1 is often mutated or inactivated in human primary lung cancers, including NSCLC ¹⁶ and small‐cell lung cancer. ¹⁷ Recent studies have shown that 10% of lung cancer patients have MEN1 deficiency. ¹⁸ Recently, it has been shown that MEN1 regulates P53 protein stability in tumor cells through methylation modifications, and that deficiency of menin results in the accumulation of DNA damage and antagonizes oncogenic Kras‐induced senescence and the epithelial‐to‐mesenchymal transition during lung tumorigenesis. ¹⁴ Furthermore, menin regulates the senescence and cytokine homeostasis of CD4⁺ T cells, and it prevents the initiation of activated CD8⁺ T cell dysfunction by restricting cellular metabolism. ¹⁹ , ²⁰ The latest research showed that cytotoxic activity and NK receptor expression were increased in menin‐deficient senescent CD8⁺ T cells. ²¹ Menin is widely involved in immunomodulation, but the role of menin in the antitumor immune of lung cancer remains to be explored.

Here, we report that AT II‐specific Men1 deficiency leads to a dramatic acceleration of Kras ^G12D‐induced lung tumorigenesis in an early stage (1–2 months), and this effect is accompanied by a dynamic upregulation of PD‐L1 and an inhibition of CD8⁺ and CD3⁺ T lymphocyte infiltration, which further attenuates the T cell‐mediated antitumor response. Mechanistically, we found that menin promotes PD‐L1 degradation by inhibiting the expression of the deubiquitinating enzyme CSN5.

2. MATERIALS AND METHODS

The details of methods are listed in Appendix S1. The materials and primers that were used in this study are listed in Tables S1 and S2.

3. RESULTS

3.1. Menin is negatively correlated with PD‐L1 in LUAD

As MEN1 was previously reported to inhibit tumor growth in human lung cancer, ¹⁶ we first analyzed the TCGA database and found that the mutation rate of MEN1 was higher in LUAD tissues than in lung squamous cell carcinoma tissues (Figure S1A). Then CCK‐8 assays were carried out to confirm the role of MEN1 in LUAD cells, including A549, NCI‐H1299, and NCI‐H1975 cells. Consistently, MEN1 overexpression inhibited the proliferation of A549, NCI‐H1299, and NCI‐H1975 cells in vitro (Figure S1B).

To investigate the potential mechanism of MEN1 in lung cancer, the previously reported KS and KMS (Men1 knockout) mouse model was used in this study. ¹⁴ However, the mouse model in this study is slightly different from previous models in the Men1 exon deletion, which might contribute to the relatively long survival time of 5 months (Figure S1C). Importantly, KMS mice show a distinctive immune microenvironment compared to KS mice, suggesting a potentially critical role of menin in regulating the immune microenvironment. ¹⁴ To initiate the development of lung carcinogenesis, mice (6–8 weeks of age) in both groups were treated with 100 mg/kg TAM i.p. (Figure S1D). The H&E staining revealed that with prolonged TAM treatment, lung tissues from KS mice predominantly displayed hyperplasia with tumor nodules, while tumors from KMS mice showed more apparent multiplicity (Figure S1E). Notably, the weight of the lung tissues from KMS mice was significantly greater (Figure S1F). Moreover, KMS mice showed significantly higher lung coefficients (lung weight/bodyweight) than KS and WT mice after 4 and 5 months of TAM treatment (Figure S1G), clearly indicating that the knockdown of Men1 accelerates the malignant progression of lung tumors. We have also validated that the lung tumors from KMS mice originate from specific Men1‐deficient AT II cells (Figure S1H).

To gain mechanistic insight into how MEN1 deletion promotes lung tumorigenesis, we undertook RNA‐seq with lung tissues from KS and KMS mice. The GO enrichment statistics showed that Men1 was positively correlated to immune‐associated pathways (Figure 1A). The GSEA of RNA‐seq data showed that immune‐related signaling pathways were downregulated in KMS lung tissues (Figure 1B–D), suggesting that Men1 knockdown in mice lung tissues may contribute to immunosuppression and escape. Considering the crucial role of PD‐L1 in maintaining an immunosuppressive microenvironment that promotes cancer progression, we investigated whether menin participates in regulating tumor immunity through PD‐L1.

FIGURE 1.

Menin deletion accelerates the progression of Kras‐induced lung adenocarcinoma. (A) Gene Ontology (GO) functional analysis of the enriched signaling pathways associated with immune‐related pathways in the lung tissue of KMS mice (n = 3) compared with KS mice (n = 3). (B–D) Gene Set Enrichment Analysis showed that regulation of the immune effector process, positive regulation of the immune effector process, and adaptive immune response pathway were enriched in KS (n = 3) compared with KMS (n = 3) lung tissues. Each of the black bars represents a gene in the pathway. (E, F) Immunohistochemical (IHC) staining for menin and programmed cell death‐ligand 1 (PD‐L1) in lung tissues of mice from each group at 1, 2, 3, 4, and 5 months (M). Scale bar, 50 μm. (G) Quantitative analysis of PD‐L1 IHC staining in (F). Data were analyzed by two‐tailed unpaired t‐tests and are presented as the mean ± SEM. **p < 0.01; ***p < P < 0.001; ****p < 0.0001. NES, normalized enrichment score.

To further investigate the relationship between menin and PD‐L1, the expression of PD‐L1 in KS and KMS mice was examined. As anticipated, upregulation of PD‐L1 was observed in lung tissues from KMS mice compared to those from KS mice (Figure 1E,F). More importantly, IHC results revealed that the upregulation of PD‐L1 in KMS mice occurred at 1–2 months; however, the high PD‐L1 expression was observed in KS mice in the third month after TAM treatment, which was markedly later than in KMS mice (Figure 1G). This suggests that deletion of Men1 in lung tumors leads to earlier accumulation of PD‐L1. Furthermore, menin may play an important role in regulating antitumor immunity.

3.2. Menin represses PD‐L1 in LUAD cell lines

To further elucidate the role of menin in the regulation of PD‐L1 expression in lung cancer, MEN1 (encodes the menin protein)/PD‐L1 overexpression plasmids were successfully transfected (Figure 2A,B). Consistent with the in vivo results (Figure 1E–G), lower expression of PD‐L1 was observed in A549 cells that stably overexpressed menin (Figure 2C). Similarly, we found PD‐L1 decreased in menin‐overexpressed NCI‐H1975 and NCI‐H1944 cells (Figure 2D). Additionally, PD‐L1 was upregulated after inhibiting the function of menin in cells by using MI‐3 (Figure 2E). To better understand the interplay between menin and PD‐L1, we tried to transiently overexpress both menin and PD‐L1 and found that PD‐L1 was significantly decreased compared with mono PD‐L1 overexpression (Figures 2F and S2A–C). However, overexpression of PD‐L1 exerted no significant effect on MEN1 (Figures 2F and S2A–C). Additionally, as shown in the western blot analysis, overexpression of menin was accompanied by dramatic inhibition of PD‐L1 in a time‐dependent manner (Figures 2G,H and S2D–G). Conversely, PD‐L1 expression was obviously increased in menin‐knockdown A549 and HEK‐293T cells (Figure 2I–K). In addition, the knockdown efficiency of MEN1 was validated by MEN1 shRNA‐resistant mutation (Figures 2I–K and S2H), suggesting that MEN1 is knocked down specifically. These findings provide evidence that menin could inhibit PD‐L1 in LUAD cells.

FIGURE 2.

Menin inhibits programmed cell death‐ligand 1 (PD‐L1) protein expression in cells. (A, B) Western blotting verified the expression of menin and PD‐L1 in A549 and HEK‐293T cells transiently transfected with the indicated constructs for 48 h. MEN1, MEN1 overexpression; PD‐L1, PD‐L1 overexpression; V, plasmid of vector, control group. (C, D) Western blot analysis of menin and PD‐L1 expression in cells. The indicated plasmids were (C) stably transfected into A549 cells or (D) transiently transfected into NCI‐H1975 and NCI‐H1944 cells. (E) Western blot analysis of PD‐L1 expression in the indicated cells. Cells were treated with DMSO or 10 μM MI‐3 for 6 h before collection. (F–H) Western blot analysis of menin and PD‐L1 expression in A549, NCI‐H1975, and HEK‐293T cells transfected with the indicated constructs at the indicated time points. M, MEN1 overexpression; M + P, MEN1 and PD‐L1 overexpression; P, PD‐L1 overexpression; V + P, vector and PD‐L1 overexpression. (I, J) RT‐quantitative PCR analysis of total MEN1 mRNA levels from control (shNC) or shMEN1 or shMEN1‐resistant (mutant MEN1). Data are presented as the mean ± SEM of three independent experiments. Statistical differences were determined using two‐tailed unpaired t‐tests. (K) Western blot assays to measure the menin and PD‐L1 levels after MEN1 knockdown or shRNA targeting MEN1 resistant (mutant MEN1). **p < 0.01; ***p < 0.001; ****p < 0.0001. ns, not significant.

3.3. Menin accelerates degradation of PD‐L1 through the Ub‐proteasome pathway

To study how menin contributes to PD‐L1 inhibition, we asked whether menin affects PD‐L1 expression by regulating its transcription. Unexpectedly, the TNMplot database showed no correlation between MEN1 and CD274 expression at the mRNA level in LUAD (Figure S2I). No significant changes were observed in the mRNA level of CD274 in either MEN1‐overexpressing or MEN1‐knockdown cells (Figures 3A–E and S2J–M). Whereas, overexpression of PD‐L1 also exerted no significant effect on the transcription of MEN1 (Figures 3A,C and S2J,L). Additionally, we verified that the mRNA levels of CD274 were not significantly different between the lung tissues of KS and KMS mice (Figure 3F,G). These findings suggested that menin suppresses PD‐L1 through a nontranscriptional regulatory pathway. Furthermore, the interaction of menin with PD‐L1 was first verified by Co‐IP assays using tagged proteins that were exogenously expressed in cells. Nevertheless, neither the endogenous nor the exogenous menin and PD‐L1 proteins interacted, as observed in vitro (Figure 3H,I).

FIGURE 3.

Menin destabilizes programmed cell death‐ligand 1 (PD‐L1) through the ubiquitin (Ub)–proteasome pathway. (A–D) RT‐quantitative PCR (qPCR) analysis of PD‐L1 and menin mRNA expression in the indicated cells transiently transfected with the indicated constructs for 24 h. Data are presented as the mean ± SEM of three independent experiments. (E–G) RT‐qPCR analysis of MEN1 and CD274 expression in the indicated cells after MEN1 knockdown (E) and whole lung tumors from KS (n = 3) and KMS (n = 3) mice at 2 and 5 months (M) after tamoxifen treatment (F, G). Statistical differences in (A–G) were determined using two‐tailed unpaired t‐tests. (H) Coimmunoprecipitation (IP) of PD‐L1 and menin in A549 and HEK‐293T cells. Endogenous PD‐L1 and menin were immunoprecipitated and analyzed by western blotting. IgG, negative control. (I) Immunoblot of immunoprecipitation and input group from the PD‐L1 pull‐down products of A549 and HEK‐293T cells transfected with the indicated constructs. (J, K) Western blot analysis of menin and PD‐L1 expression in A549 and HEK‐293T cells after transfection for 24 h. Cells were treated with 25 μM MG132 at the indicated time points. (L, M) Protein stability analysis of PD‐L1 in the indicated cells after transfection for 24 h. The cells were treated with 20 μg/mL cycloheximide (CHX) at the indicated time points before collection. (N, O) Quantification of PD‐L1 protein levels in (L, M). Data are presented as the mean ± SEM of three independent experiments. *p < 0.05; **p < 0.01; ***p < 0.001. M, MEN1 overexpression; M + P, MEN1 and PD‐L1 overexpression; ns, not significant; P, PD‐L1 overexpression; V + P, vector and PD‐L1 overexpression.

Excluding transcriptional and direct modulation, we considered whether menin affects PD‐L1 levels by regulating its protein stability. Next, the cells were treated with the proteasome inhibitor MG132 and the protein translation inhibitor CHX. Compared to the control cells, menin overexpression did not inhibit the protein synthesis of PD‐L1 after MG132 treatment (Figure 3J,K). However, overexpression of MEN1 significantly shortened the half‐life of the PD‐L1 protein, suggesting that menin destabilized PD‐L1 by the Ub–proteasome pathway (Figures 3L–O and S3A–D). Collectively, these results supported the conclusion that the Ub–proteasome pathway, rather than the transcriptional regulation or direct interaction, contributes to menin‐mediated PD‐L1 degradation.

3.4. Menin promotes PD‐L1 degradation by inhibiting expression of DUB CSN5

Ubiquitin molecules are covalently attached to substrate proteins through an enzyme cascade that is catalyzed by E1 activating enzymes, E2 conjugating enzymes, and E3 ligating enzymes to regulate the proteasomal degradation of these substrates. ²² Deubiquitination is an essential regulatory step in the Ub‐dependent pathway, and DUBs mediate the removal of Ub moieties from substrate proteins. ²³ We determined whether menin inhibited PD‐L1 stabilization by catalyzing its ubiquitination. The Co‐IP assays indicated that menin promoted the polyubiquitination of PD‐L1 in A549 and HEK‐293T cells (Figures 4A and S4A,B). To identify the intermediate regulators of menin‐mediated PD‐L1 ubiquitination, the mRNA levels of E3 Ub ligases (SPOP, ²⁴ , ²⁵ STUB1, ²⁶ FBXO22, ²⁷ NEDD4, ²⁸ and CBLB ²⁹ ) and DUBs (COPS5, ⁸ , ³⁰ USP22, ³¹ , ³² and USP9X ³³ ) that target PD‐L1 were examined by RT‐qPCR after MEN1 overexpression (Figure S4C–P). Ultimately, we found that CSN5 (encoded by the COPS5 gene) showed significant changes in both the MEN1 overexpression and knockdown groups (Figure 4B–E), and this protein is known to modulate the deubiquitination and stabilization of PD‐L1. ³ Additionally, analysis of lung cancer data from TNMplot revealed a negative correlation between MEN1 expression and COPS5 expression (Figure 4F).

FIGURE 4.

Menin inhibits the expression of the deubiquitinating enzyme COP9 signalosome subunit 5 (CSN5) and inhibits its interaction with programmed cell death‐ligand 1 (PD‐L1). (A) Ubiquitination assay of PD‐L1 in response to MEN1 overexpression. A549 and HEK‐293T cells were transiently transfected with the indicated constructs and treated with 25 μM MG132 for 6 h prior to ubiquitination analysis. Ubiquitinated PD‐L1 was subjected to western blot analysis with an anti‐ubiquitin (Ub) Ab. (B–E) RT‐quantitative PCR analysis of CSN5 mRNA expression in A549 and HEK‐293T cells after overexpression or knockdown of MEN1. Statistical differences were determined using two‐tailed unpaired t‐tests. (F) Scatter plots showing correlations between MEN1 and COPS5 mRNA expression in lung cancer. Data were derived from TNMplot, with data from NCBI‐GEO and TCGA. Two‐tailed Spearman's correlation test. (G) Western blot analysis of CSN5 expression in MEN1‐overexpressing and MEN1‐knockdown cells. (H, I) Protein stability of PD‐L1 in A549 and HEK‐293T cells after transfection for 48 h. Cells were treated with 20 μg/mL cycloheximide (CHX) at the indicated time points and analyzed by western blotting. (J) Coimmunoprecipitation of immunoprecipitates (IP) and input from HA pull‐down products derived from A549 and HEK‐293T cells transfected and treated as in (A) but with the additional transfection of CSN5 overexpression plasmid. *p < 0.05; **p < 0.01.

Consistent with the changes in transcription, the CSN5 protein was also suppressed by MEN1 overexpression (Figure 4G). Conversely, CSN5 was upregulated after MEN1 knockdown (Figure S4Q). Half‐life analysis indicated that overexpression of menin decreased PD‐L1 and shortened its protein half‐life by suppressing the deubiquitinating enzyme CSN5, thus further promoting PD‐L1 degradation (Figure S4R and Figure 4H). In addition, CSN5 combined with menin overexpression or knockdown reversed the effect of menin on PD‐L1 (Figure S4S and Figure 4H,I). Moreover, the Co‐IP assay suggested that MEN1 overexpression attenuated the direct interaction of CSN5 and PD‐L1, thereby promoting the ubiquitination of PD‐L1 (Figure 4J). According to these results, we showed that menin promotes the Ub‐mediated degradation of PD‐L1 by inhibiting the expression of the deubiquitinating enzyme CSN5.

3.5. Menin deficiency inhibits T cell infiltration and function

The major types of immune cells in the TME include CD8⁺ T cells, CD3⁺ T cells, CD4⁺ T cells, Tregs, and NK cells. ³⁴ Moreover, the main subtypes of T cells are CD8⁺ and CD4⁺ T cells, which are engaged in T cell‐mediated cytotoxicity and regulate the immune response. ³⁴ , ³⁵ Overexpression of PD‐L1 on the surface of tumor cells leads to immune escape by promoting the apoptosis and exhaustion of T cells. ³⁶ The above results indicated that menin can destabilize PD‐L1 by the Ub–proteasome pathway. Therefore, it is important to explore whether menin deficiency activates the PD‐L1/PD‐1 axis and blocks the activation and cytotoxicity of T cells. First, the gene expression profiles of KS and KMS mice lung tissues were compared using RNA‐seq to further investigate the role of menin in T cell activation. Notably, heatmaps showed that deletion of the Men1 gene was associated with downregulation of genes related to T cell activity and chemokine activity (Figure 5A). Consistent with this finding, the gene pathway enrichment analysis and GSEA of RNA‐seq showed that the downregulated genes in the lung tissue of KMS mice were significantly enriched in some signaling pathways associated with T cell function (Figure 5B–E). The most enriched pathway was the chemokine activity (GO:0008009) pathway based on the enrichment score (Figure 5F). Further RT‐qPCR results showed that the expression of proinflammatory cytokines (including IFN‐γ, TNF‐α, and Il‐1β) and T cell‐derived chemokines that are associated with immune cell recruitment (including Ccl5, Cxcl9, and Cxcl10) ³⁷ , ³⁸ was decreased in the lung tissues from KMS mice (Figure 5G–L). Consistently, RT‐qPCR and IHC results proved that the knockdown of Men1 suppressed the production of the lytic molecule granzyme B (a marker of T cell activation) (Figure 5M–O). To further delineate the alterations in the TME following Men1 deletion, we analyzed the TILs in blood cells and lung tumor tissues from KS and KMS mice using FACS assays. In line with this result, FACS showed lower percentages of blood lymphocyte and tumor‐infiltrating TNF‐α/CD3⁺CD8⁺ T cells and IFN‐γ/CD3⁺CD8⁺ T cells in KMS mice compared to KS mice (Figures 5P,Q and S5A,B).

FIGURE 5.

Menin deficiency reduces T cell infiltration. (A) Hierarchical clustering of significantly altered genes in KMS (n = 3) lung tissues compared to KS (n = 3) lung tissues. (B) Scatter plot showing the results of Gene Ontology (GO) biological process enrichment analysis of the significantly downregulated genes after Men1 knockdown. The DAVID GO pathway identifiers are noted. (C–E) Gene Seat Enrichment Analysis showed that CCR chemokine receptor binding, chemokine activity, and chemokine‐mediated signaling pathway were negatively associated with the lung tissue of KMS mice. Each of the black bars represents a gene in the pathway. (F) Relevant differential genes involved in chemokine‐activated pathways were significantly downregulated in KMS lung tissues compared to KS lung tissues. (G–M) RT‐quantitative PCR analysis of the gene expression of IFN‐γ, TNF‐α, Il‐1β, Ccl5, Cxcl9, Cxcl10, and Gzmb in whole lung tissues from KS (n = 3) and KMS (n = 3) model mice at 2 months after tamoxifen treatment. (N) Immunohistochemical (IHC) staining for granzyme B (GZMB) in lung tissues of KS and KMS mice from each group at 1, 2, 3, 4, and 5 months. Scale bar, 50 μm. (O) Quantitative analysis of GZMB IHC staining in the lung tissues of KS and KMS mice in each month (M) in (N). (P) Exemplifying the gating strategy of flow cytometry analysis. Forward versus side scatter (FSC vs. SSC) gating was used to identify cells and exclude cell debris. Fluorescence‐activated cell scorting (FACS) analysis of representative images of the percentage of γ‐interferon (IFN‐γ) and tumor necrosis factor‐α (TNF‐α) positivity in CD3⁺CD8⁺ subpopulations in blood lymphocyte of KS and KMS mice. (Q) FACS analysis of representative images of the percentage of IFN‐γ and TNF‐α positivity in CD3⁺CD8⁺ subpopulations in lung tumors tissues of KS and KMS mice. Source data are provided as source data files. *p < 0.05; **p < 0.01; ***p < 0.001; ns, not significant. NES, normalized enrichment score; TPM, transcripts per million.

Next, based on the RNA‐seq results, we reasoned that MEN1 deletion might affect TME in tumors. We used TIMER 2.0 to evaluate the association between MEN1 mRNA level and major immune cell subtypes by various algorithms. The gene expression analysis revealed a strong positive correlation between the expression of MEN1 and the infiltration of CD4⁺ T cells (central memory and Th1 cells), macrophages (M0), and NK cells (Figure S5C,D). Then, IHC results showed that knockdown of Men1 resulted in an earlier upregulation of PD‐L1 and exhaustion of CD8⁺, CD3⁺, and CD4⁺ T lymphocytes during the progressive stage of mouse lung tumors (Figures 1G and S5E–J). This observation indicated that menin deficiency leads to an attenuated antitumor immune response by stabilizing PD‐L1 and further inhibits T cell function by inhibiting CD8⁺ T cell‐mediated and CD3⁺ T cell‐mediated cytotoxicity in lung tissues.

3.6. Menin deficiency contributes to poor prognosis of LUAD by repressing tumor immunity

To investigate the clinical relevance of our results, we randomly collected 38 human LUAD specimens and undertook IHC staining for menin, PD‐L1, CD8, CD3, and CD4 (Figure 6A). We found that 18.42% (7/38) of the samples had low menin expression (menin‐low) and that menin levels were negatively correlated with PD‐L1 abundance in 38 LUAD samples (Figure 6B,C). Moreover, it was observed that individuals in the menin‐high group almost overlapped with those in the CD3 and CD4 positive groups (Figure 6D). The IHC staining analysis revealed a positive correlation between menin expression and CD3⁺ and CD4⁺ T cell infiltration in LUAD tissues (Figure 6E). However, in contrast to the results of the mouse lung tissue experiments, there was no significant correlation between menin expression and CD8⁺ T cell infiltration (Figure 6F,G). This difference can probably be attributed to the heterogeneity of human LUAD tissues. These findings similarly confirmed that loss of menin function can upregulate PD‐L1 and facilitate the immune escape of tumors.

FIGURE 6.

Menin could be a potential target for tumor immunotherapy. (A) Immunohistochemistry (IHC) was carried out to identify target proteins in clinical lung adenocarcinoma (LUAD) specimens (n = 38). Menin‐high and menin‐low suggest that menin expression in tumor tissues is similar to or lower than that in paraneoplastic tissues, respectively. “Menin‐high” describes a menin‐positive area ≥ 50,000 in a single field of view; “Menin‐low” describes a positive area < 50,000. Representative images of IHC staining for the protein expression of the immune escape marker programmed cell death‐ligand 1 (PD‐L1) and the T cell infiltration markers CD8, CD3, and CD4 in human lung cancer samples. Scale bar, 50 μm. (B) Bar chart represents the proportion of high and low menin expression in 38 human LUAD specimens. (C–G) Venn diagrams show the number of samples with high PD‐L1, CD3, CD4, and/or CD8 expression in menin‐high and menin‐low LUAD samples. Correlation analysis between menin expression and PD‐L1, CD3, CD4, and CD8 expression was undertaken by analysis of the Spearman correlation coefficient (r value indicated, two‐tailed). For each of the 38 samples, three images were randomly captured. IHC scores were assessed by Image‐Pro Plus. Scale bar, 50 μm. (H) Association of the MEN1 to CD274 mRNA expression level ratio (MEN1 as numerator and CD274 as denominator) with overall survival in LUAD datasets from the KM‐plotter (http://kmplot.com/analysis/). These patients (n = 672) were diagnosed with LUAD and were not restricted to stage, grade, gender, smoking history, or treatment. (I) Kaplan–Meier survival rates were estimated using KM‐plotter showing the effect of MEN1 gene expression on overall survival (left, n = 459) and relapse‐free survival (right, n = 138) of anti‐PD‐L1‐treated patients from the time of diagnosis. These patients are not restricted to tumor type or gender. Log‐rank (Mantel–Cox) test. Number at risk: number of people in follow‐up at each time point. HR, hazard ratio.

We wondered whether the ratio of MEN1 to CD274 could be a clinical marker for determining survival in LUAD patients. The KM plotter revealed that a low ratio of MEN1/CD274 was associated with short overall survival of patients with LUAD (Figure 6H). Menin‐inhibited PD‐L1 deubiquitination could contribute to suppressing tumor immune escape. Therefore, we attempted to evaluate the potential of menin to be used in anti‐PD‐L1 immunotherapy and found that high expression of MEN1 was associated with high overall survival (Figure 6I, left) and relapse‐free survival (Figure 6I, right) of patients who received anti‐PD‐L1 treatment. This could reveal a synergistic effect by which high expression of menin cooperates with anti‐PD‐L1 treatment to promote antitumor immunity by decreasing PD‐L1 expression and inducing T cell‐mediated cytotoxicity. Taken together, these results indicated that targeting menin might be a potential strategy for enhancing the effect of immunotherapy on lung cancers.

4. DISCUSSION

This study focused on menin and found the dynamic relationship between menin and PD‐L1 expression in Kras‐induced LUAD mice and cell lines. The MEN1 KO mice showed that menin deficiency alters lung TME in mice, leading to premature exhaustion of CD8⁺, CD3⁺, and CD4⁺ T lymphocytes. Finally, we explored the mechanism such that menin can downregulate PD‐L1 by inhibiting the deubiquitinating enzyme CSN5 (Figure 7).

FIGURE 7.

Proposed model of menin‐mediated programmed cell death‐ligand 1 (PD‐L1) regulation. Menin deletion accelerates the progression of Kras‐induced lung adenocarcinoma, deregulates PD‐L1, and alters the lung tumor microenvironment in mice, which further leads to premature exhaustion of CD8⁺, CD3⁺, and CD4⁺ T lymphocytes. Mechanistically, menin promotes PD‐L1 destabilization and degradation by inhibiting expression of COP9 signalosome subunit 5 (CSN5). PD‐1, programmed cell death‐1; Ub, ubiquitin.

As a ubiquitously expressed nuclear protein, menin acts as a scaffold or adaptor protein to regulate gene transcription and cell signaling in multiple biological pathways. ³⁹ Menin protein was discovered to play a role in ubiquitination modifications. ⁴⁰ In addition, menin can regulate and activate immune cells. ²⁰ Despite the low mutation rate of menin in LUAD, we cannot exclude other reasons that contribute to the low expression of menin in lung tissues and thus promote the malignant progression of lung cancer.

Recent studies have indicated different PTM mechanisms that regulate PD‐L1 protein expression, including glycosylation, ubiquitination, phosphorylation, and acetylation. ⁴¹ However, the signaling pathways that govern the deubiquitination and accumulation of PD‐1/PD‐L1 in lung tumors are not yet completely understood. Accumulating evidence has shown that CSN5 possesses deubiquitinating activity, and it plays an important role in the development and progression of cancer. ³ For example, a previous study reported that CSN5 facilitates lung cancer cell growth through stabilizing survival. ⁴² Also, CSN5 reduces PD‐L1 ubiquitination, stabilizes PD‐L1, and promotes immune evasion. ⁶ , ⁹ In this study, our data provide evidence that menin promotes the ubiquitination and degradation of PD‐L1 by inhibiting the expression of the deubiquitinating enzyme CSN5, thereby decreasing the stability of PD‐L1 in cells.

CTLA4 and PD‐L1/PD‐1 are usually activated in TME and repress T cell‐mediated cytotoxicity. ⁴³ , ⁴⁴ Then, the function of T cells diminishes over time. ⁴⁵ Hence, elucidating the mechanisms underlying immune escape is crucial for understanding the survival and progression of lung cancer. Patient data from TCGA showed that the expression level of MEN1 is negatively correlated with neutrophil and CD8⁺ T cell tumor infiltration in lung cancer. ⁴⁶ Our findings in mice reveal that Men1 deficiency accelerates the progression of Kras‐induced LUAD and enhances PD‐L1 expression. Moreover, the decrease in chemokine activity after Men1 deletion may imply that the infiltration of immune cells secreting chemokines is inhibited. These effects are accompanied by decreasing numbers of CD8⁺ T cells, which promotes the immune evasion and proliferation of tumor cells. Whereas, our cellular results reveal that menin plays an important role in preventing T cell exhaustion by inhibiting the stability of PD‐L1, and menin can effectively contribute to immune regulation during the early development of lung cancer.

Moreover, we noted a negative correlation between the protein levels of menin and PD‐L1 in some human lung tumors. This could lead to an effective antitumor adaptive immune response in lung cancer development. Therefore, we supposed that most tumors with low MEN1 expression inhibit CD8⁺ T cell chemotaxis, recruitment, and killing activity to evade immune surveillance. This result may be beneficial for some patients who are resistant to anti‐PD‐L1 mAb therapies.

We assume that the activation of immune checkpoints is necessary for cancer cells to evade the surveillance and killing by T cells, allowing cancer cell survival in cancer development, as the number of tumor cells is low. As tumor cells proliferate and accumulate, the T cell response becomes overwhelmed, reducing the need for further immune checkpoint activation. In this study, we found that PD‐L1 was upregulated in KMS mouse lung tumors compared to KS mouse lung tumors. A previous study showed that Kras can inhibit menin expression by promoting DNA methylations. ¹⁰ Therefore, we inferred that Kras mutation may indirectly upregulate PD‐L1 by inhibiting menin expression in the lung tissues of KS mice. In contrast, knocking down menin in the lung tissues of KMS mice resulted in a more pronounced decrease in menin expression, which subsequently resulted in a loss of function to inhibit PD‐L1.

Overall, these findings suggested that PD‐L1 levels are prematurely upregulated in the Men1‐deficient mouse model, which subsequently results in the immune evasion and progression of lung cancer. This could explain why high PD‐L1 expression (tumor cells with PD‐L1 expression ≥50%) is associated with improved responses to ICIs ⁴⁷ and may be influenced by the abundance of menin and the lung tumor stage. This could also explain the heterogeneity of PD‐L1 expression and the inconsistent sensitivity of the same type of lung cancer to PD‐L1‐targeting therapy. However, it is still a challenge to identify which subset of patients will be responsive to ICI therapies.

AUTHOR CONTRIBUTIONS

Cuncun Zhang: Conceptualization; data curation; validation; writing – original draft. Ningning Sun: Conceptualization; data curation; validation; writing – original draft. Qingze Fei: Data curation; validation. Linlin Peng: Data curation; validation. Chengyu Wei: Validation; visualization. Xiangyu Liu: Validation; visualization. Sainan Miao: Data curation; validation. Mengqi Chai: Data curation; validation. Fang Wang: Formal analysis; resources. Di Wang: Methodology; resources; supervision. Jingfang Hong: Methodology; resources; supervision. Shenghai Huang: Methodology; resources; supervision. Shihao Zhang: Conceptualization; methodology; supervision; visualization; writing – review and editing. Huan Qiu: Conceptualization; funding acquisition; methodology; project administration; supervision; visualization; writing – review and editing.

FUNDING INFORMATION

This work was supported by the National Natural Science Foundation of China (82002450, to H.Q.), Research Program for Higher Education Institutions in Anhui Province (2023AH050656, to H.Q.), Basic and Clinical Collaboration Enhancement Program of Anhui Medical University (2020xkjT023, to H.Q.), Postgraduate Innovation Research and Practice Program of Anhui Medical University (YJS20230064, to C.C.Z.), and 2023 Postgraduate Youth Training Program of School of Nursing, Anhui Medical University (hlqm12023015, to N.N.S.).

CONFLICT OF INTEREST STATEMENT

The authors declare no conflict of interest.

ETHICS STATEMENTS

Approval of the research protocol by an institutional review board: Approved by the Ethics Review Committee of Anhui Medical University.

Informed consent: Informed consent was obtained from all patients before the study.

Registry and the registration no. of the study/trial: N/A.

Animal studies: Approved by the Ethics Review Committee of Anhui Medical University.

Supporting information

Appendix S1.

Zhang C, Sun N, Fei Q, et al. MEN1 deficiency stabilizes PD‐L1 and promotes tumor immune evasion of lung cancer. Cancer Sci. 2024;115:2515‐2527. doi: 10.1111/cas.16196

DATA AVAILABILITY STATEMENT

The data analyzed in Figures S1A, S2I, 4F, S5C,D, and 6H,I were obtained from TIMER 2.0 ⁴⁸ (http://timer.comp‐genomics.org/timer/), TNMplot ⁴⁹ (https://tnmplot.com/analysis/), KM‐plotter ⁵⁰ (http://kmplot.com/analysis/), and TCGA ⁵¹ (https://tcga‐data.nci.nih.gov/tcga/). Other original data that support the findings of this study are available from the corresponding author upon reasonable request.