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Journal for Immunotherapy of Cancer logoLink to Journal for Immunotherapy of Cancer
. 2025 Jun 23;13(6):e010151. doi: 10.1136/jitc-2024-010151

Hypoxia upregulates the expression of PD-L1 via NPM1 in breast cancer

Yihui Yu 1,2,0,1, Ran Sun 3,0,1, Feiyun Hu 2, Zite Ding 2, Xin Li 2, Jiyuan Han 1,2, Leiting Liang 2, Tian Wang 2, Guifu Xi 2, Xueyi Dong 1,2, Yanlei Li 1,2, Xiulan Zhao 1,2,*, Danfang Zhang 1,2,✉
PMCID: PMC12186055  PMID: 40550561

Abstract

Background

A hypoxic microenvironment is the most frequent characteristic in tumor microenvironment. Programmed death-ligand 1 (PD-L1) is an important molecule and therapeutic target that mediates the immune response of tumor cells. Previous studies have shown that hypoxia can lead to increased expression of Nucleophosmin 1 (NPM1) and PD-L1. However, the exact regulatory mechanisms of NPM1 and PD-L1 expression under hypoxic conditions are still poorly understood.

Methods

The relationships among hypoxia, NPM1 and PD-L1 were explored by western blotting, immunofluorescence staining, flow cytometry and chromatin immunoprecipitation-quantitativePCR(ChIP-qPCR). Animal tumor models were established to explore the effect of NPM1 expression on tumor growth. The relationships between NPM1 and breast cancer (BC) clinical features and immune infiltration were revealed by bioinformatics analysis.

Results

NPM1 mediates increased PD-L1 expression in the hypoxic microenvironment of BC. HIF-1α can increase the expression of NPM1 by activating the p-AKT pathway and binding to the NPM1 promoter. Increased expression of NPM1 can promote tumor growth and inhibit T cell infiltration. Bioinformatics analysis showed that the high expression of NPM1 was associated with poorer survival and immunosuppression in patients with BC.

Conclusions

The hypoxic microenvironment promotes PD-L1 expression via NPM1 in BC, which may be further associated with the inhibition of tumor immunity. NPM1 may serve as a potential target for modulating PD-L1 immunotherapy.

Keywords: Breast cancer, Hypoxia, Immunotherapy, NPM1, PD-L1

WHAT IS ALREADY KNOWN ON THIS TOPIC

  • Programmed death-ligand 1 (PD-L1) is a crucial immune checkpoint molecule and therapeutic target that regulates tumor immune evasion. The hypoxic tumor microenvironment (TME) can upregulate PD-L1 expression. Nucleophosmin 1 (NPM1), as a multifunctional protein, plays a role in cellular adaptation to hypoxia and modulates PD-L1 expression.

WHAT THIS STUDY ADDS

  • In breast cancer (BC), the hypoxic microenvironment upregulates NPM1 expression through HIF-1α-mediated activation of the p-AKT pathway and direct binding to the NPM1 promoter, consequently enhancing PD-L1 expression on tumor cells. Elevated NPM1 expression promotes tumor progression while suppressing T cell infiltration. NPM1 may serve as a prognostic biomarker in patients with BC and is associated with an immunosuppressive TME.

HOW THIS STUDY MIGHT AFFECT RESEARCH, PRACTICE OR POLICY

  • Our findings provide novel mechanistic insights into hypoxia-induced PD-L1 expression. NPM1 emerges as a potential key mediator of immunosuppression in BC within hypoxic microenvironments.

Background

As the most common malignant tumor in women worldwide, breast cancer (BC) seriously endangers women’s health.1 Because of its high incidence and complex pathogenesis, it is critical to find effective therapeutic targets for BC.2 3 BC is recognized as a heterogeneous disease whose occurrence and development are easily regulated by the tumor microenvironment (TME).4 During the course of tumor progression, owing to the rapid proliferation of cells and insufficient blood supply, the internal area of the tumor is often hypoxic.5 The hypoxic microenvironment has been shown to be an important stimulant of tumor angiogenesis, maintenance of tumor stem cell activity, cellular immune escape, and tumor cell invasion and metastasis.5,7

Immunotherapy has become an effective treatment for BC in recent years.8,10 As an important immune checkpoint molecule and a target for immunotherapy, programmed death-ligand 1 (PD-L1) expressed on tumor cells can interact with programmed cell death protein 1 (PD1) on infiltrating lymphocytes in the TME to weaken the effector T-cell response, thereby protecting tumor cells from immune attack.11 High expression of PD-L1 is positively correlated with tumor size, tumor grade, and lymph node metastasis in BC.12 Moreover, hypoxia can significantly increase the expression of PD-L1 on macrophages, dendritic cells, and tumor cells in a HIF-1α-dependent manner.13 Thus, deep insight into the regulatory mechanism of the hypoxic microenvironment and PD-L1 is urgently needed.

Nucleophosmin 1 (NPM1) is a nucleolar protein that shuttles between the nucleus and cytoplasm during the cell cycle.14 Research has shown that NPM1 is involved in many biological processes such as ribosome biogenesis, cell mitosis, DNA repair, cell replication and transcription.15 There is evidence that the immune function of CD8+ T cells in patients with acute myeloid leukemia (AML) with NPM1 mutations is decreased, and that the recognition of tumor cell antigens and other immune markers by immunogenic T cells is also decreased.16 17 In lung adenocarcinoma (LUAD), NPM1 expression is negatively correlated with B cells, CD4+ T cells and macrophages.18 More studies have shown that NPM1 mutations induce higher levels of PD-L1 expression.19 These findings suggest that NPM1 plays an important role in cancer immune regulation, but the mechanism has not been fully characterized.

In this study, we revealed that a hypoxic microenvironment can increase the expression of PD-L1 via NPM1. Moreover, HIF-1α promotes NPM1 expression by activating the p-AKT pathway and binding to the NPM1 promoter. Our study reveals the role of NPM1 in regulating PD-L1 expression in the hypoxic microenvironment of BC. NPM1 may be a potential target for affecting PD-L1 immunotherapy in BC.

Methods

Cell culture

The human BC cell lines MDA-MB-231 and MCF-7 were purchased from Shanghai Fuheng Biotechnology. All the cells were grown in Dulbecco’s Modified Eagle’s Medium (DMEM) supplemented with 10% fetal bovine serum (FBS) (Invitrogen, California, USA) and 1% penicillin/streptomycin. The hypoxic microenvironment was induced by treating the cells with a final concentration of 150 µM cobalt chloride (CoCl2) solution for 48 hours. All the cells were cultured in an incubator containing 5% CO2 at 37℃.

Plasmids and transfection

The plasmids were synthesized by GeneCopoeia, including NPM1 complementary cDNA (EX-G0147-Lv122), a negative control (EX-NEG-Lv122), NPM1 small interfering RNAs (CS-HSH102948-LVRU6GP-01), SH-Control (CSHCTR001- LVRU6GP), HIF-1α small interfering RNAs (HSH008831-LVRH1MH) and SH-Control (CSHCTR001-LVRH1MH). The pReceiver-Lv122 vector was used to clone the open reading frame(ORF) sequence targeting NPM1. The psi-LVRU6GP vector was used to clone the SH-RNAs targeting NPM1. The sequence of SH-NPM1 was 5 ′ - GGAATGTTATGATAGGACA-3′. The psi-LVRH1MH vector was used to clone the SH-RNAs targeting HIF-1α. The sequence of SH- HIF-1α was 5 ′ GCCGAGGAAGAACTATGAACA-3 ′ . Cell transfection was performed using lentivirus packaging according to the instructions (Lenti-Pac HIV Expression Kit, GeneCopoeia). The HEK293T cells were used to collect the supernatant of the virus. The virus was then concentrated and transfected into BC cells with polybrene. Transfected cells were selected with appropriate concentrations of puromycin for at least 1 week. Cells cotransfected with HIF-1α and NPM1 plasmids were stably transfected with the HIF-1α plasmid in a cell line that was stably transfected with the NPM1 plasmid.

Western blot assay

Protein was extracted from cells with RIPA lysis buffer (P0013B, Beyotime). The nuclear protein was extracted according to the instructions (KGB5302-100, KeyGEN BioTech). Proteins were transferred to polyvinylidene fluoride (PVDF) membranes using polyacrylamide gel electrophoresis. The membrane was blocked with 5% skim milk for 1 hour and then incubated with primary antibody overnight at 4°C. The following antibodies were used: Carbonic anhydrase 9 (CA9) (11 071–1-AP, Proteintech, 1:1000), NPM1 (60 096–1-Ig, Proteintech, 1:5000), PD-L1 (66 248–1-Ig, Proteintech, 1:2000), HIF-1α (20 960–1-AP, Proteintech, 1:500), Tubulin (66 031–1-Ig, Proteintech, 1:2000), AKT (AF6259, Affinity, 1:500), phospho-AKT (S473) (4060S, Cell Signaling, 1:500). They were then incubated at room temperature with secondary antibody for 2 hours. The protein bands were obtained by C-Digit Blot Scanner (Gene Company) and analyzed with ImageJ software.

Immunofluorescence staining assay

The cells were fixed with cold methanol for 20 min, permeabilized with 1% Triton X- 100 for 30 min, and then blocked with 5% FBS for 30 min. Primary antibodies against NPM1 (60 096–1-Ig, Proteintech, 1:200), and PD-L1 (66 248–1-Ig, Proteintech, 1:200) from Proteintech were added to the samples, which were subsequently incubated overnight at 4°C. The cell nuclei were stained with 4′,6-diamidino-2-phenylindole (Sigma). Images were obtained using fluorescence microscopy.

Flow cytometry assay

The cells were harvested and washed two times with phosphate-buffered saline (PBS) for 5 min each. A total of 1 × 106 cells were added to 100 ul of flow cytometry staining buffer (Proteintech, PF00018). PD-L1 antibody (Proteintech, APC-65081) was added and the samples were incubated at 4°C for 20–40 min in the dark. The cells were resuspended in 200–500 ul of staining buffer and analyzed via C6 (BD Biosciences) flow cytometry.

ChIP-qPCR assay

The MDA-MB-231 cells were crosslinked with 1% formaldehyde for 10 min at 37℃. The reaction was stopped by the addition of glycine solution. An ultrasonic crusher was used to obtain DNA fragments no larger than 2000 bp in size. Chromatin was immunoprecipitated with either control IgG (Ab171870, 5 µg) or the HIF-1α (20 960–1-AP; Proteintech; 5 µg) or NPM1 (60 096–1-Ig, Proteintech;5 µg) primary antibody. After washing and DNA purification, PCR-ready samples are obtained. PCR was performed using real‐time PCR Master Mix (TB Green) according to the manufacturer’s instructions (Takara, RR820A). The sequences of primers used are listed below: NPM1 (primer 1: forward 5′-CTTAGGGCGATGTCCTTGCT-3′, reverse 5′-AGTTACCGGCCAGACTTACG-3′; primer 2: forward 5′- TAAGAGCGGCAAGAAGTCAGAG-3′, reverse 5′- GGAAAGATGTAGTTACCGGCCA-3′; primer 3: forward 5′- GCAAGAAGTCAGAGTCGGC-3′, reverse 5′-GGGCCAAAAGGGGACTGAATC-3′; primer 4: forward 5′-GTTCTTACAAGTCACCCGCTTTC-3′, reverse 5′- TCGCCCTAAGAGAGCTCGG-3′). The primer sequences of PD-L1 were listed below: primer 1, forward 5′-CTTCGAAACTCTTCCCGGTG-3′, reverse 5′-ACC TCTGCCCAAGGCAGCAA-3′; primer 2, forward 5′-AAACCAAAGCCATATGGGTC-3′, reverse 5′-AGCCAACATCTGAACGCACC-3′; primer 3, forward 5′-TAGAATAGGCTTCCGCAGCC-3′, reverse 5′-CTAGAAAGTAGGTGTGTGTG-3′.

Dual-luciferase reporter assay

HEK293T cells were seeded in 24-well plates and transfected with 1 µg/well of the NPM1 promoter plasmid and the SH-HIF-1α plasmid (0.4, 0.8, or 1.2 µg). After 3 hours, 1 mL of culture medium containing 30% FBS was added to the cell culture medium, and the mixture was incubated for an additional 24 hours. Then, the medium was replaced with fresh medium, and the mixture was cultured for 24 hours. Then, the supernatant was collected with a 0.45 µm filter. Luciferase activity was detected via a Secerte-Pair dual luminescence assay kit from GeneCopoeia according to the manufacturer’s instructions.

Transwell assay

Migration assays were performed in 24-well plates. Serum-free medium and BC cells (1×105) were added to the upper chamber, and DMEM supplemented with 10% FBS was added to the lower chamber. After incubation for 48 hours, the cells were fixed with cold methanol and then stained with crystal violet. The migrated cells were counted manually under the microscope. For the invasion assays, the transwell chambers were coated with Matrigel 1 day in advance, and the other steps were the same as those for the migration assays.

3D Matrigel culture assay

Matrigel diluted 1:3 with DMEM was added to the bottom of the 24-well plate, which was then placed in an incubator to dry for use. The BC cell suspension was added to a 24-well plate, and the number of vasculogenic mimicry (VM) tubes was observed under a microscope after 48 hours at 37℃.

Xenograft animal models

Tientsin albino 2 (TA2) female mice aged 4–6 weeks, which constitute a mouse model of spontaneous BC, were provided by the Animal Center of Tianjin Medical University. All steps were carefully administered to protect the welfare of the animals and to minimize suffering. All animals were maintained under a specific pathogen-free grade vivarium. All animal experiments have complied with the Animals in Research: Reporting In Vivo Experiments guidelines.20 We collected the breast tumors and prepared them into cell suspension. The cancer cells (5×106 cells) were then injected under the skin of the TA2 mice (n=12). The injected mice were randomly divided into two groups of 6 mice. When the tumor diameter reached 0.5 cm, SH-NPM1 plasmids (n=6, 6.25 µg) and control plasmids (n=6, 6.25 µg) were injected into the tumors every 2 days along with the RNA transfection reagent (Entransterin vivo, Engreen). We measured tumor volume (length × width2/2) and plotted growth curves. After five injections, the mice were sacrificed simultaneously.

Immunohistochemical staining assay

The tissue sections were dewaxed and rehydrated in a series of graded alcohols. First, 3% H2O2 was used to block endogenous peroxidase, followed by microwave antigen repair. After the tissue sections were naturally restored to room temperature, they were blocked with 10% goat serum (ZSGB-Bio) for 30 min. The membranes were incubated with primary antibody at 4℃ overnight, after which they were incubated with secondary antibody. The following primary antibodies were used: NPM1 (60 096–1-Ig, Proteintech), PD-L1 (66 248–1-Ig, Proteintech), and CD3 (17 617–1-AP, Proteintech). All the sections were then subjected to diaminobenzidine (DAB) staining, followed by hematoxylin staining. PBS was used to replace the primary antibody in the negative control test. The staining intensity and proportion of stained cells were scored as follows: 0 (negative), 1 (weak), 2 (medium) and 3 (high); 0 (negative), 1 (≤25%), 2 (25–50%), and 3 (>50%). The score of the section was finally determined by the above two scores.

Bioinformatics analysis

The Kaplan-Meier plotter (Kaplan-Meier plotter kmplot.com) was used to analyze the effects of NPM1 expression on the relapse-free survival (RFS) and overall survival (OS) of patients with different subtypes of BC. The relationships between NPM1 expression and the clinical features of patient with BC were analyzed by UALCAN (The University of ALabama at Birmingham CANcer data analysis Portal) (https://ualcan.path.uab.edu/). The TIMER (Tumor IMmune Estimation Resource) (https://cistrome.shinyapps.io/timer/) database was used to analyze the relationships between NPM1/CA9 and the immune infiltration of BC. The difference in NPM1 expression across the abundances of tumor-infiltrating lymphocytes was analyzed using the TISIDB (Tumor-Immune System Interaction Database) (TISIDB hku.hk). The Cancer Genome Atlas breast cancer (TCGA-BRCA) dataset was downloaded. R package “bioR22.cor.R” was used to analyze the associations between NPM1 and PD-L1 expression and immune depletion molecules.

Statistical analysis

Statistical analysis was performed using GraphPad Prism V.8.3.0 software. The mean difference between two groups was compared using Student’s t-test, and the mean difference between multiple groups was compared using one-way analysis of variance. P values of less than 0.05 were considered statistically significant. All the data are representative of three independent experiments.

Results

Hypoxia promotes the expression of PD-L1 in breast cancer

The ability of CoCl2 to induce a hypoxic cellular microenvironment has been well described.21 In accordance with previous experiments, CoCl2 at a final concentration of 150 µM was ultimately selected for use in this study to induce a hypoxic microenvironment in cells. CA9 protein expression was detected to verify that induction of the hypoxic microenvironment was successful (online supplemental figure 1).

Next, we examined the changes in PD-L1 expression in BC cells induced by hypoxia. As shown in figure 1A,B, western blot analysis revealed that the expression of PD-L1 in MDA-MB-231 and MCF-7 cells was significantly increased after the induction of hypoxia, and the content of PD-L1 in MDA-MB-231 cells was higher than that in MCF-7 cells. Consistent results were also obtained by cell immunofluorescence staining (figure 1C,D).

Figure 1. PD-L1 expression is increased in the hypoxic microenvironment of breast cancer. (A–D) Western blot (A, B) and immunofluorescence (C, D) analyses of the expression of PD-L1 in MDA-MB-231 and MCF-7 cells treated with or without CoCl2 (150 µM) for 48 hours. P values were calculated using Student’s t-test. (*p<0.05, **p<0.01, ***p<0.001, ****p<0.0001, Bar=200 µm). CoCl2, cobalt chloride; DAPI, 4′,6-diamidino-2-phenylindole; H, hypoxia; N, normoxia; PD-L1, programmed death-ligand 1.

Figure 1

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NPM1 mediates the increase in PD-L1 expression in the hypoxic microenvironment

On the basis of our previous analysis of hypoxia-dependent spatial transcriptomics, we found that, as a hypoxia-related gene, NPM1 has the same spatial distribution characteristics as PD-L1 (figure 2A). Therefore, we further studied the regulatory relationship between NPM1 and PD-L1.

Figure 2. (A) Spatial feature plots of HIF1A, CA9, NPM1, and PD-L1 in samples and their gene expression in different groups.0, Invasive tumor; 1, Adaptive survival tumor; 5, Necrosis periphery; 6, Hypoxic tumor; 10, Necrosis center. (B, C) Western blot analysis of background NPM1 expression in MDA-MB-231 and MCF-7 cells. (D, E) Immunofluorescence analysis of the fluorescence intensity of NPM1 in MDA-MB-231 and MCF-7 cells. (F, G) The transfection efficiency of the NPM1 knockdown and overexpression plasmids was measured by western blotting in MDA-MB-231 and MCF-7 cells. P values were calculated using Student’s t-test. (***p<0.001, ****p<0.0001, Bar=200 µm). CA9, Carbonic anhydrase 9; DAPI, 4′,6-diamidino-2-phenylindole; NPM1, Nucleophosmin 1; PD-L1, programmed death-ligand 1; ST, Spatial transcriptome.

Figure 2

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First, the background expression of NPM1 in BC cells was detected by western blotting (figure 2B,C) and immunofluorescence staining (figure 2D,E). The results revealed that the expression level of NPM1 was significantly higher in MDA-MB-231 cells than in MCF-7 cells. Next, stably transfected MDA-MB-231 SH-NPM1 and MCF-7 EX-NPM1 cell lines were constructed via the transfection of lentiviral packaged plasmids. Figure 2F,G shows the cell transfection efficiency.

Next, we investigated the influence of changes in NPM1 expression on PD-L1 in hypoxia. Figure 3A–D shows that the expression of NPM1 and PD-L1 in MDA-MB- 231 and MCF-7 cells increased after the induction of hypoxia. The expression of PD-L1 increased after upregulation of NPM1 expression in MCF-7 cells, and decreased after downregulation of NPM1 expression in MDA-MB-231 cells. After hypoxia was induced in MDA-MB-231 SH-NPM1 cells, the expression of NPM1 increased compared with that in the normoxia group, and PD-L1 also showed the same trend. In MCF-7 EX-NPM1 cells, NPM1 and PD-L1 expression was significantly increased in the hypoxia group compared with that in the normoxia group. Moreover, our cell immunofluorescence staining results were consistent with the western blot results, as shown in figure 3E–H. These findings suggest that the hypoxic microenvironment of BC positively regulates PD-L1 expression by promoting NPM1 expression. The flow cytometry results confirmed that the increase in NPM1 expression in the hypoxic microenvironment promoted the expression of PD-L1 on the MDA-MB-231 and MCF-7 cell surfaces (online supplemental figure 2).

Figure 3. NPM1 mediates increased PD-L1 expression in the hypoxic microenvironment of breast cancer. (A–D) Western blot analysis of NPM1 and PD-L1 expression in MDA-MB-231 SH-Control, MDA-MB-231 SH-NPM1, MCF-7 EX-Control and MCF-7 EX-NPM1 cells with or without CoCl2 (150 µM, 48 hours). (E–H) Immunofluorescence analysis of NPM1 and PD-L1 fluorescence intensity in MDA-MB-231 SH-Control, MDA-MB-231 SH-NPM1, MCF-7 EX-Control and MCF-7 EX-NPM1 cells with or without CoCl2 (150 µM, 48 hours). The mean difference between two groups was compared using Student’s t-test, and the mean difference between multiple groups was compared using one-way ANOVA. (*p<0.05, **p<0.01, ***p<0.001, ****p<0.0001, Bar=200 µm). ANOVA, analysis of variance; CoCl2, cobalt chloride; DAPI, 4′,6-diamidino-2-phenylindole; H, hypoxia; N, normoxia; NPM1, Nucleophosmin 1; PD-L1, programmed death-ligand 1.

Figure 3

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Hypoxia promotes NPM1 expression through HIF-1α-mediated activation of the p-AKT pathway and binding to the NPM1 promoter region

To investigate whether the hypoxic microenvironment affects NPM1 and PD-L1 expression through p-AKT, we examined the effects of the hypoxic microenvironment on p-AKT levels, NPM1 and PD-L1 expression in MDA-MB-231 and MCF-7 cells. The results revealed that the expression of HIF-1α, p-AKT, NPM1 and PD-L1 increased in hypoxia (figure 4A,B). When HIF-1α was knocked down in normoxia, the expression of p-AKT, NPM1 and PD-L1 did not significantly change (figure 4A,B). Moreover, after hypoxia of MDA-MB-231 SH-HIF-1α and MCF-7 SH-HIF-1α cells, the expressions of HIF-1α, p-AKT, NPM1 and PD-L1 were increased compared with the normoxia group, but decreased compared with MDA-MB-231 SH-Control and MCF-7 SH-Control hypoxic groups. These findings indicate that HIF-1α is involved in the regulation of NPM1 and PD-L1 in the hypoxic microenvironment.

Figure 4. The hypoxic microenvironment of breast cancer regulates the expression of NPM1 and PD-L1 through the activation of the p-AKT pathway by HIF-1α. (A, B) Western blot analysis of HIF-1α, p-AKT, NPM1 and PD-L1 expression in MDA-MB- 231 SH-Control, MDA-MB-231 SH-HIF-1α, MCF-7 SH-Control and MCF-7 SH-HIF-1α cells with or without CoCl2 (150 µM, 48 hours). (C, D) Western blot analysis of the p-AKT, NPM1 and PD-L1 expression in MDA-MB-231 cells treated with MK-2206 (0 µM, 2 µM, 4 µM and 8 µM) for 48 hours. (E, F) Western blot analysis of the p-AKT, NPM1 and PD-L1 expression in MDA-MB-231 cells treated with or without MK-2206 (8 µM) and CoCl2 (150 µM) for 48 hours. The mean difference between two groups was compared using Student’s t-test, and the mean difference between multiple groups was compared using one-way ANOVA. (*p<0.05, **p<0.01, ***p<0.001, ****p<0.0001). ANOVA, analysis of variance; CoCl2, cobalt chloride; H, hypoxia; N, normoxia; NPM1, Nucleophosmin 1; PD-L1, programmed death-ligand 1.

Figure 4

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To further investigate the regulation of NPM1 and PD-L1 by p-AKT, MDA-MB-231 cells were treated with a p-AKT inhibitor (MK-2206). As shown in figure 4C,D, the level of p-AKT decreased significantly, and the expression of NPM1 and PD-L1 also decreased gradually with increasing p-AKT inhibitor concentration. We then treated MDA-MB-231 cells with the most effective concentration (8 µM) of MK-2206. The results showed that the expression of NPM1 and PD-L1 increased in the hypoxic group, but the increase in NPM1 and PD-L1 expression was reversed after the addition of the p-AKT inhibitor in the hypoxic group (figure 4E,F). This finding provides strong evidence for the regulation of NPM1 and PD-L1 expression by activating the p-AKT pathway in the hypoxic microenvironment.

The HIF-1α plasmid and NPM1 plasmid were transfected into MCF-7 and MDA-MB-231 cells to further investigate the role of HIF-1α in the process by which hypoxia promotes NPM1 and PD-L1 expressions. The results revealed that the expression of NPM1 and PD-L1 was significantly decreased when the level of HIF-1α was knocked down in hypoxia (figure 5A,B). Furthermore, the expression of PD-L1 was increased by inducing hypoxia in MCF-7 cells transfected with SH-HIF-1α and EX-NPM1 plasmids. The level of PD-L1 in MDA-MB-231 cells transfected with SH-HIF-1α and SH-NPM1 plasmids was further reduced (figure 5A,B). These findings suggest that the hypoxic microenvironment further regulates PD-L1 expression by promoting NPM1 expression through HIF-1α. Furthermore, we designed primers for the NPM1 promoter region (figure 5C). Through the chromatin immunoprecipitation-quantitativePCR(ChIP-qPCR) assay, we found that NPM1 was highly enriched in primers 1, 2 and 4 (figure 5D). These findings indicate that HIF-1α can bind to the NPM1 promoter region. We then constructed an NPM1 promoter plasmid and used dual-luciferase reporter assay to verify the effect of HIF-1α on NPM1 promoter activity. Figure 5E,F shows that NPM1 promoter activity decreased gradually with increasing HIF-1α down-regulation. These results indicate that HIF-1α positively regulates NPM1 promoter activity. Moreover, through a ChIP-qPCR assay, we demonstrated that NPM1 can bind to the promoter region of PD-L1 (online supplemental figure 3).

Figure 5. HIF-1α binds the NPM1 promoter region to promote NPM1 expression in a hypoxic microenvironment. (A, B) Western blot analysis of HIF-1α, NPM1 and PD-L1 expression in MDA-MB-231 and MCF-7 cells with or without transfection of the HIF-1α knockdown plasmid and the NPM1 plasmid. All the cells were treated with CoCl2 (150 µM) for 48 hours. The cells cotransfected with NPM1 and HIF-1α plasmids were transfected with the HIF-1α plasmid on the basis of stable transfection of the NPM1 plasmid. (C, D) ChIP-qPCR analysis of the regions where HIF-1α binds to the NPM1 promoters in MDA-MB-231 cells. (E, F) Dual luciferase reporter gene assay analysis of the effect of HIF-1α knockdown on the activity of the NPM1 promoter in MDA-MB-231 cells. The SH-HIF-1α plasmid was added to 0.4 µg, 0.8 µg or 1.2 µg respectively. The mean difference between two groups was compared using Student’s t-test, and the mean difference between multiple groups was compared using one-way ANOVA. (*p<0.05, **p<0.01, ***p<0.001, ****p<0.0001). ANOVA, analysis of variance; CoCl2, cobalt chloride; ChIP, chromatin immunoprecipitation; IgG, immunoglobulin G; IP, input; mRNA, messenger RNA; NPM1, Nucleophosmin 1; PD-L1, programmed death-ligand 1; qPCR, quantitative PCR; SEAP, secreted alkaline phos-phatase.

Figure 5

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Knockdown of NPM1 suppresses tumor growth in vivo

To determine the role of NPM1 in tumor growth in vivo, we constructed a TA2 mouse xenograft model. The tumor growth curve (figure 6A) revealed that the tumor growth rate decreased after the injection of the SH-NPM1 plasmid compared with that in the control group. Moreover, we demonstrated through transwell assays that increased NPM1 expression in a hypoxic group can enhance the invasion and migration ability of BC cells (online supplemental figure 4A,B). Similarly, consistent results were observed in the three-dimensional Matrigel culture assay (online supplemental figure 4C). These results reveal that the hypoxic microenvironment promotes the expression of NPM1, and subsequently enhances the degree of malignancy of BC cells.

Figure 6. Knockdown of NPM1 suppresses tumor growth in vivo. (A) A total of 5×106 breast tumor cells was subcutaneously injected into the TA2 mice (4–6 weeks, n=12). Mice carrying tumors were randomly divided into two groups. When the tumor diameter reached 0.5 cm, NPM1 knockdown plasmids (n=6, 6.25 µg) and control plasmids (n=6, 6.25 µg) were injected into the tumor every 2 days. The mice were killed after five injections, and tumor growth curves were measured and plotted. (B) Immunohistochemical results of NPM1, PD-L1, and CD3 in tumors in NPM1 knockdown and its control group. P values were calculated using Student’s t-test. (*p<0.05, **p<0.01, Bar=100 µm). NPM1, Nucleophosmin 1; PD-L1, programmed death-ligand 1; TA2, Tientsin albino 2.

Figure 6

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Immunohistochemical staining of mouse tumor tissue revealed that the expression of NPM1 and PD-L1 decreased in tumors injected with the SH-NPM1 plasmid, whereas the expression of CD3 in T cells increased (Figure 6B). Therefore, we speculate that NPM1 can inhibit tumor immunity by promoting the expression of PD-L1.

High expression of NPM1 is associated with the clinical features and immune infiltration of BC

The Kaplan-Meier plotter analysis revealed that high expression of NPM1 was associated with poor RFS in patients with BC and BC subtypes (basal-like), but there was no significant correlation in luminal A and luminal B patients. Moreover, NPM1 expression showed no significant association with OS in patients with BC and BC subtypes (figure 7A). Further analysis of the UALCAN database revealed that NPM1 was more highly expressed in breast tumor tissues than in normal tissues (figure 7B). The expression of NPM1 was higher in young patients with BC (21–40 years) and was positively correlated with lymph node metastasis (figure 7B). Next, TIMER analysis revealed that NPM1 was negatively correlated with CD4+ T cells in BRCA (figure 8A). Moreover, CA9, a highly expressed molecule in hypoxic solid tumors, was also negatively correlated with the infiltration of some immune cells in BRCA (figure 8B). TISIDB analysis also revealed that NPM1 was significantly negatively correlated with CD4, CD8, Treg and natural killer T (NKT) cells (figure 8C). We further analyzed the relationship between NPM1 expression and classical immunomodulatory molecules by downloading TCGA data. We found that NPM1 was positively correlated with lymphocyte-activation gene 3 (LAG3) and cytotoxic T-lymphocyte associated protein 4 (CTLA4), and that the correlation with LAG3 was statistically significant (figure 8D). PD-L1 was significantly positively correlated with LAG3, B-lymphocyte and T-lymphocyte attenuator, and CTLA4.

Figure 7. Bioinformatics analysis of the correlation between NPM1 and the clinical features of patient with breast cancer. (A) Effects of NPM1 expression on the RFS and OS of patient with breast cancer and its different subtypes according to the Kaplan-Meier plotter database. (B) NPM1 transcription in subgroups of patients with BRCA, stratified on the basis of age and lymph node metastasis (UALCAN).(*p<0.05, **p<0.01, ***p<0.001, ****p<0.0001). BRCA, breast invasive carcinoma; NPM1, Nucleophosmin 1; OS, overall survival; RFS, relapse-free survival; UALCAN, The University of ALabama at Birmingham CANcer data analysis Portal.

Figure 7

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Figure 8. (A) NPM1 expression is correlated with tumor purity and the level of immune infiltration in BRCA (TIMER). (B) Relationships between CA9 and immune infiltration in BRCA (TIMER). (C) Relationships between NPM1 expression and the abundance of tumor-infiltrating lymphocytes (TILs) in BRCA (TISIDB). (D) Analysis of the correlation between NPM1 and PD-L1 expression and the expression of immunomodulatory molecules in breast cancer. (*p<0.05, **p<0.01, ***p<0.001, ****p<0.0001). BTLA, B and T-lymphocyte attenuator; BRCA, breast invasive carcinoma; CA9, Carbonic anhydrase 9; CTLA4, cytotoxic T-lymphocyte associated protein 4; LAG3, lymphocyte-activation gene 3; NKT, natural killer T; NPM1, Nucleophosmin 1; PD-L1, programmed death-ligand 1; TIMER, Tumor IMmune Estimation Resource; TISIDB, Tumor-Immune System Interaction Database.

Figure 8

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Discussion

The TME plays an important role in dynamically regulating cancer progression and influencing treatment.22 23 The hypoxic microenvironment is one of the more common types of microenvironments, which can make tumor cells more aggressive.24 Moreover, studies have shown that the hypoxic microenvironment can enhance the ability of tumor cells to resist immune attack and evade immune surveillance.25 Our results revealed that a hypoxic microenvironment led to a significant increase in PD-L1 expression in BC cells. PD-L1, a key molecule in the immune escape of tumor cells, binds to PD1 on T cells to cause dysfunction and decrease the activity of T cells, thereby preventing cytotoxic T cells from effectively targeting tumor cells.24 26 27 Moreover, there is evidence that the expression of PD-L1 on melanoma cells and non-small cell lung cancer cells is significantly increased after culture in a hypoxic microenvironment.13

Through our previous spatial transcriptome analysis,28 we found that NPM1 was spatially correlated with hypoxia and PD-L1 . Li et al reported that NPM1 protein levels in normal human lymphocytes and fibroblasts increased significantly after 6 hours of hypoxia. In HEK293 cells, the hypoxic microenvironment can increase the transcription of the NPM1 gene and promote its expression.29 Our results show that NPM1 mediates the increase in PD-L1 expression in the hypoxic microenvironment. Moreover, the ChIP-qPCR assay revealed that NPM1 can bind to the promoter region of PD-L1 (online supplemental file 3). Our results are consistent with those reported by Ge et al that NPM1 can specifically bind to the PD-L1 promoter and activate its transcription in triple-negative breast cancer.30 Liu et al reported that NPM1 is involved in the regulation of immune invasion in LUAD and can be used as a prognostic marker.18 In AML, the inhibition of NPM1 mutations suppresses the immune function of CD8+ T cells.16 Therefore, our results indicate that NPM1 may regulate tumor immune infiltration through PD-L1 in BC.

Next, we focused on the regulatory mechanism between hypoxia and NPM1. NPM1 is a multifunctional protein whose N-terminal region contains 16 potential phosphorylation sites. Phosphorylation can affect the structure and stability of the N-terminal region, and thus affect the subcellular localization and corresponding functions of NPM1.31 AKT is a cytoplasmic signal transduction protein connected to a variety of signal transduction pathways, and p-AKT is its activated form.32,35 A study in mouse motor neuron cells revealed a significant increase in p-AKT levels after two hours of hypoxia.36 In our study, hypoxia can enhance the expression of p-AKT, NPM1, and PD-L1 through HIF-1α. Moreover, the results show that the increase in NPM1 and PD-L1 expression caused by the hypoxic microenvironment was reversed after the addition of p-AKT specific inhibitors. These results demonstrate that the increased expression of NPM1 and PD-L1 in the hypoxic microenvironment of BC is achieved through AKT activation by HIF-1α. Further study illustrates that hypoxia regulates the expression of NPM1 through HIF-1α, and then regulates the expression of PD-L1 in BC cells. Moreover, we also found that HIF-1α can bind the NPM1 promoter region and positively regulate the activity of the NPM1 promoter. Koukoulas et al reported that the silencing of NPM1 expression in HeLa cells under hypoxia significantly reduced the transcriptional activity of HIF-1.37 These findings indicate that there is an interaction between HIF-1 and NPM1. Our experimental results revealed that the expression of HIF-1α increased when the expression of NPM1 increased. These results suggest that HIF-1α and NPM1 may be mutually regulated through the p-AKT pathway. The regulatory mechanism between HIF-1α and NPM1 still needs to be further explored. In previous studies, NPM1 was recognized as both a tumor suppressor and a proto-oncogene during tumorigenesis.38,40 In this study, we demonstrated that NPM1 can promote tumor growth in vivo. Meanwhile, increased NPM1 expression improved the metastasis and tube formation ability of BC cells (online supplemental file 4A–C). Our results provide new evidence that NPM1 accelerates tumor progression.

Finally, we described the relationships between NPM1 and clinical features and some immune molecules in BC. We found that high expression of NPM1 led to poor RFS in patients with BC, but the difference in OS was not statistically significant. Moreover, high NPM1 expression was significantly associated with RFS in patients with the basal-like subtype of BC. These results suggest that NPM1 may be more important in influencing the prognosis of basal-like BC. In addition, UALCAN database analysis revealed that the expression of NPM1 was positively correlated with age and lymph node metastasis in patients with BC. These results provide evidence that NPM1 is a poor prognostic indicator of BC. We then analyzed the associations between NPM1 and immune molecules. We found that NPM1 and CA9 expression was negatively correlated with CD4+ T cell infiltration levels in BRCA. Further analysis of TCGA data also revealed that NPM1 and PD-L1 were significantly positively correlated with LAG3, which has been shown to have negative regulatory effects on T cells.41 Moreover, immunohistochemical staining of mouse tumor tissues revealed that knockdown of NPM1 inhibited PD-L1 expression, and T cell infiltration was promoted. Previous studies have shown that overexpression of NPM1 can inhibit the infiltration of B cells and NK cells in LUAD.18 A negative correlation between NPM1 and immune infiltration was also found in Ewing sarcoma.42

Kreon et al reported that NPM1, as a key molecule that can interact with HIF-1α, is critical for cellular adaptation to hypoxia.37 Xin et al reported that NPM1 promotes tumor immune escape by inhibiting IRF1-mediated antigens and reprogramming the immunosuppressive TME.43 Our experimental results and bioinformatics analysis provide a basis for the idea that the hypoxic microenvironment of BC may regulate the expression of PD-L1 through NPM1, thus inhibiting cellular immune infiltration.

Our results demonstrate that NPM1 plays a key role in immunosuppression induced by the hypoxic microenvironment in BC. NPM1 may be a potential therapeutic molecule in immunotherapy of BC.

Conclusion

In the hypoxic microenvironment, HIF-1α activates the p-AKT pathway and increases NPM1 promoter activity to upregulate NPM1 expression, thereby promoting PD-L1 expression in BC cells (figure 9). Our study provides a new rationale for the increase in PD-L1 expression in the hypoxic microenvironment. NPM1 may be a poor prognostic indicator in patients with BC and inhibit BC immune infiltration. Based on the regulatory effect of NPM1 on PD-L1, NPM1 may be a potential target affecting PD-L1 immunotherapy, and its role in clinical work still needs further verification.

Figure 9. Mechanistic diagram of the hypoxic microenvironment regulating PD-L1 expression in tumor cells (by Figdraw). The hypoxic microenvironment promoted the expression of NPM1 through HIF-1α, and then promoted the expression of PD-L1 on tumor cells. NPM1, Nucleophosmin 1; PD-1, programmed cell death protein 1; PD-L1, programmed death-ligand 1.

Figure 9

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Supplementary material

online supplemental file 1
jitc-13-6-s001.jpg (754KB, jpg)
DOI: 10.1136/jitc-2024-010151
online supplemental file 2
jitc-13-6-s002.jpg (1.4MB, jpg)
DOI: 10.1136/jitc-2024-010151
online supplemental file 3
jitc-13-6-s003.jpg (501.7KB, jpg)
DOI: 10.1136/jitc-2024-010151
online supplemental file 4
jitc-13-6-s004.jpg (2.6MB, jpg)
DOI: 10.1136/jitc-2024-010151

Acknowledgements

We gratefully acknowledge the funding supporter, and the support provided by the Department of Pathology at Tianjin Medical University General Hospital and the Department of Pathology at Tianjin Medical University.

Footnotes

Funding: This study was funded by the project of the National Nature Science Foundation of China (No. 82373278), and the Scientific Research Plan Project Fund of Tianjin Municipal Education Commission (Grant No.2023KJ056).

Provenance and peer review: Not commissioned; externally peer reviewed.

Patient consent for publication: Not applicable.

Ethics approval: The animal experiments involved in the article were approved by the Laboratory Animal Management and Use Committee (IACUC) of Tianjin Medical University (TMUaMEC2023127).

Data availability free text: All data relevant to the study are included in the article or uploaded as supplementary information. The TCGA breast cancer RNA expression data is available in the TCGA database (https://portal.gdc.cancer.gov). The spatial transcriptome profiling data are available in online repositories. The names of the repository/repositories and accession number(s) can be found below: 10.6084/m9.figshare.21695735.v1.

Data availability statement

All data relevant to the study are included in the article or uploaded as supplementary information.

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Associated Data

This section collects any data citations, data availability statements, or supplementary materials included in this article.

Supplementary Materials

online supplemental file 1
jitc-13-6-s001.jpg (754KB, jpg)
DOI: 10.1136/jitc-2024-010151
online supplemental file 2
jitc-13-6-s002.jpg (1.4MB, jpg)
DOI: 10.1136/jitc-2024-010151
online supplemental file 3
jitc-13-6-s003.jpg (501.7KB, jpg)
DOI: 10.1136/jitc-2024-010151
online supplemental file 4
jitc-13-6-s004.jpg (2.6MB, jpg)
DOI: 10.1136/jitc-2024-010151

Data Availability Statement

All data relevant to the study are included in the article or uploaded as supplementary information.

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