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. 2025 Dec 12;28:14. doi: 10.1186/s13058-025-02193-5

Nuclear PD-L1 drives IFN-γ-promoted lung metastasis of triple-negative breast cancer via POLR2A-mediated transcriptional activation of LY6E

Xu Wang 1,2,3,#, Qi Zhou 1,2,3,#, Pu Wang 1,3,5,#, Shunshun Bao 1,2,3, Xianzheng Wei 1,2,3, Xuan Zhao 1,2,3, Rui Hou 4, Sijin Li 1,2,3, Zhangchun Guan 1,2,3, Wen Ma 1,2,3, Junnian Zheng 2,3,✉, Dan Liu 1,2,3,✉, Ming Shi 1,2,3,✉
PMCID: PMC12817839  PMID: 41388312

​Abstract

Background

​​Triple-negative breast cancer (TNBC) patients exhibiting high PD-L1 expression demonstrate poor responses to anti-PD-L1 therapy and aggressive lung metastasis. The paradoxical role of PD-L1 beyond its immune checkpoint function and the impact of interferon-γ-secreted during immunotherapy-on metastasis remain poorly understood.

Methods

Integrated reanalysis of single-cell RNA sequencing (scRNA-seq) data from TNBC lung metastases identified enriched signaling pathways. IFN-γ function was assessed using murine and human TNBC cell lines, employing in vitro assays and in vivo modeling in both immunocompetent and immunodeficient mice. CRISPR/Cas9-mediated PD-L1 ablation, pharmacological inhibitors, RNA sequencing (RNA-seq), chromatin immunoprecipitation sequencing (ChIP-seq), co-immunoprecipitation (Co-IP), bioinformatics analyses, and in vivo metastasis assays were utilized to dissect underlying mechanisms.

Results

scRNA-seq revealed significant enrichment of IFN-γ signaling within a distinct metastatic TNBC cluster. IFN-γ pretreatment potently enhanced the lung metastatic capacity of TNBC cells in both immunocompetent and immunodeficient murine models. CRISPR/Cas9-mediated PD-L1 ablation abolished IFN-γ-driven metastasis without affecting proliferation, indicating an immune checkpoint-independent mechanism. Mechanistically, IFN-γ facilitated HDAC2-mediated deacetylation of PD-L1, promoting its nuclear translocation. RNA-seq identified lymphocyte antigen 6 complex locus E (LY6E) as a key downstream effector, with expression correlating with PD-L1 in TNBC patient samples. Nuclear PD-L1 bound to the RNA polymerase II subunit POLR2A to form a transcriptional complex that directly activated LY6E expression, thereby driving metastatic dissemination.

Conclusion

Our findings unveil a novel IFN-γ-nuclear PD-L1/POLR2A-LY6E signaling axis critical for TNBC lung metastasis. This immune-independent mechanism, driven by nuclear PD-L1 transcriptional activity, provides a mechanistic basis for the limited efficacy of anti-PD-L1 antibodies against metastasis and nominates nuclear PD-L1 complexes and LY6E as potential therapeutic targets to overcome metastatic resistance in TNBC.

Supplementary Information

The online version contains supplementary material available at 10.1186/s13058-025-02193-5.

​Keywords: Triple-negative breast cancer, IFN-γ, PD-L1 nuclear translocation, POLR2A, LY6E, Lung metastasis, Immune checkpoint-independent mechanism, Therapeutic resistance

​​Background

Triple-negative breast cancer (TNBC), defined by the absence of estrogen receptor (ER), progesterone receptor (PR), and HER2 amplification, represents the most aggressive and metastasis-prone breast cancer subtype, accounting for 15–20% of all breast cancers yet contributing disproportionately to mortality due to limited therapeutic options [1]​​. ​​Studies reveal that 36.9% of TNBC patients develop lung metastases—a rate significantly higher than that in other subtypes—and these metastases drive rapid disease progression despite standard chemotherapy​​ [2]. ​​While immune checkpoint inhibitors targeting PD-1/PD-L1 have emerged as promising therapies, recent clinical trials have reported disappointing objective response rates of only ~ 10% in metastatic TNBC, with over 60% of PD-L1-positive patients still experiencing progressive lung metastasis following anti-PD-L1 monotherapy [3–5]​​. ​​This stark clinical paradox highlights a critical gap in understanding PD-L1 biology: conventional immune checkpoint blockade fails to address potential non-canonical, metastasis-driving functions of PD-L1 that may operate independently of T-cell regulation.​​

The tumor microenvironment following immunotherapy provides mechanistic clues to this conundrum. ​​Upon PD-L1 blockade, reactivated T cells secrete high levels of IFN-γ—a pleiotropic cytokine with context-dependent roles in cancer progression [6, 7]​​. ​​Although IFN-γ enhances antigen presentation and tumor immunogenicity, it simultaneously upregulates immunosuppressive molecules, including PD-L1 itself, creating a self-reinforcing resistance loop​​ [8]. ​​Moreover, emerging evidence suggests that IFN-γ may directly promote metastatic dissemination in certain contexts: low-dose IFN-γ enhances cancer stemness and lung colonization capacity in non-small cell lung cancer (NSCLC), while chronic IFN-γ exposure accelerates metastasis in TNBC models through undefined mechanisms [9, 10]​​. ​​These observations raise a pivotal hypothesis: Could IFN-γ paradoxically fuel TNBC metastasis through non-immune functions of PD-L1?​​

This hypothesis gains credence from recent breakthroughs revealing PD-L1’s non-canonical roles. Beyond its membrane-bound immune checkpoint function, PD-L1 translocates to the nucleus where it regulates DNA damage repair, modulates chromatin dynamics, and forms transcription factor complexes to drive oncogenic programs—mechanisms entirely independent of PD-1 binding [11–16]​​. Studies have shown that nuclear PD-L1 can promote the progression of lung adenocarcinoma, bladder cancer, hepatocellular carcinoma, and melanoma [17–23]​​. ​​However, whether such mechanisms operate in TNBC metastasis, particularly under IFN-γ stimulation, remains unexplored—a significant knowledge gap given TNBC’s unique metastatic tropism for the lung.​​

Here, we identify a novel IFN-γ-PD-L1/POLR2A-LY6E signaling axis that drives lung metastasis in TNBC through immune checkpoint-independent mechanisms. ​​We demonstrate that IFN-γ promotes HDAC2-mediated PD-L1 deacetylation and nuclear translocation, where PD-L1 physically associates with the RNA polymerase II subunit POLR2A to form a transcriptional complex. This complex selectively activates LY6E-a glycosylphosphatidylinositol-anchored protein known to potentiate TGF-β/Smad signaling and lung metastatic dissemination [24–26]​​. ​​Our work not only explains the limited efficacy of anti-PD-L1 antibodies against metastasis but also provides the first evidence that targeting nuclear PD-L1 complexes may overcome therapeutic resistance in TNBC.​

Materials and methods​​

Cell culture and reagents​​

The mouse triple-negative breast cancer (TNBC) cell line 4T1 (ATCC® CRL-2539™), and human TNBC cell line MDA-MB-231 (ATCC® HTB-26™) were cultured in DMEM (Sigma) supplemented with 10% fetal bovine serum (FBS; TransGen) and maintained at 37 °C under 5% CO₂. Mouse triple-negative breast cancer (TNBC) cell line EMT6 was cultured in 1640 (Sigma) supplemented with 10% fetal bovine serum. Recombinant murine IFN-γ (Peprotech, 315-05) and human IFN-γ (Peprotech, 300-02) were dissolved in sterile PBS. The HDAC2 inhibitor Santacruzamate A (MCE, HY-15616), importin-α inhibitor Ivermectin (MCE, HY-15310), and STAT3 inhibitor S3I-201 (Selleck, S1155) were dissolved in DMSO.

Flow cytometry for PD-L1 surface expression​​

4T1 cells treated with IFN-γ (0-100 ng/mL; Peprotech #315-05) for 24 h were harvested and stained with an anti-mouse PD-L1-PE antibody (1:100; BioLegend #124307, clone 10F.9G2) for 30 min at 4 °C. Data were acquired on BD FACSCanto™ II and analyzed using ​​FlowJo v10.8.1​​.

​​Cell proliferation assay (CCK-8)​​

4T1 cells (1 × 104cells/well) were seeded in 96-well plates and treated with IFN-γ (0-100 ng/mL) for 24 h. Cell viability was assessed using ​​CCK-8 reagent​​ (VICMED, VC5001L) according to the manufacturer’s protocol. Absorbance was measured at 450 nm using a Biotek Cytation 5 instrument after 1 h of incubation.

​​CRISPR/Cas9-mediated PD-L1 knockout​​

PD-L1 knockout (PD-L1 KO) 4T1 cells were generated using CRISPR/Cas9 as previously described. Briefly, sgRNAs targeting Pdcd1 (encoding PD-L1) were cloned into lentiCRISPRv2 (Addgene #52961). Lentivirus was produced in HEK293T cells and transduced into 4T1 cells. Transduced cells were selected with 4 µg/mL puromycin (VICMED). Knockout efficiency was validated via Western blotting and flow cytometry using an anti-PD-L1 antibody (Cell Signaling Technology, #60475).

Transwell migration and invasion assays​​

PD-L1 wild-type (WT)/knockout (KO) 4T1 cells (8 × 104 cells) in serum-free DMEM were seeded into Transwell inserts (8-µm pore size; Corning #3422). For invasion assays, inserts were pre-coated with ​​Matrigel​​ (100 µg/mL; Bio-Techne BME001-05). After 24 h (migration) or 48 h (invasion), cells were fixed with 4% paraformaldehyde (PFA), stained with 0.1% crystal violet (Servicebio G1014-50ML), and imaged under a ​​Nikon Eclipse Ti2 microscope​​. Migrated and invaded cells were quantified using ​​ImageJ v1.53​​.

Colony formation assay​​

PD-L1 WT/KO 4T1 cells (500/well) were seeded in 6-well plates and cultured for 7 days. Colonies were fixed with 4% PFA, stained with 0.1% crystal violet, and counted using an automated colony counter (​​GelCount, Oxford Optronix​​). Colonies with a diameter > 50 μm were included.

​​​​Western blotting and subcellular fractionation​​

Whole-cell lysates were extracted using RIPA buffer (Beyotime, P0013B) containing protease and phosphatase inhibitors (Roche, 11697498001). Nuclear/cytoplasmic fractions were isolated using the Nuclear and Cytoplasmic Protein Extraction Kit (Beyotime, P0027). Proteins (20–30 µg) were separated by SDS-PAGE, transferred to nitrocellulose (NC) membranes, and probed with primary antibodies against PD-L1 (CST, #13684; #60475), POLR2A (CST, #2629), LY6E (Proteintech, 22144-1-AP), Claudin-1 (Proteintech, 13050-1-AP), STAT1 and phosphorylated STAT1 (CST, #14994; #9167), and GAPDH (Proteintech, 60004-1-Ig). HRP-conjugated secondary antibodies (CST) and ECL reagent (PerkinElmer) were used for detection.

Immunofluorescence for PD-L1 nuclear localization​​

MDA-MB-231 cells grown on coverslips were treated with IFN-γ (50 ng/mL) for 48 h, fixed with 4% paraformaldehyde (PFA), permeabilized with 0.1% Triton X-100, and blocked with 5% bovine serum albumin (BSA). Cells were incubated overnight at 4 °C with anti-PD-L1 antibody (1:200; CST #13684) followed by Alexa Fluor 555-conjugated secondary antibody (1:500; Thermo Fisher # A-21428). Nuclei were counterstained with DAPI (1 µg/mL; Beyotime C1002). Images were acquired on a ​​Zeiss LSM 900 confocal microscope​​. PD-L1 nuclear translocation was quantified using ​​ImageJ v1.53​​.

​​RNA sequencing and qPCR​​

Total RNA was extracted using TRIzol (Takara, 9109) and reverse-transcribed with PrimeScript RT Kit (Takara, RR037A). qPCR was performed using ChamQ SYBR Color qPCR Master Mix (Vazyme, Q411-02) on a Roche LightCycler 96 instrument. For RNA-seq, total RNA isolated from tumor cells was subjected to library preparation using oligo(dT) magnetic beads (Thermo Fisher, #25-61005) for mRNA enrichment. Libraries were constructed via magnesium-mediated fragmentation (NEB, #E6150S), cDNA synthesis, and end repair, yielding 300 ± 50 bp fragments [27]. PE150 sequencing was performed on Illumina Novaseq™ 6000 (LC Bio). Raw reads were processed to obtain FPKM values, and differential expression was analyzed in R (v4.3.2) using DESeq2 and edgeR with the criteria of |Log₂FC| ≥ 1.5 and adjusted p-value (adj. p) < 0.05. The RNA sequencing matrix data are provided in Supplementary Table 1.

​​In vivo lung metastasis models​​

All animal experiments were approved by the Animal Ethics Committee of Xuzhou Medical University (Approval No.: 202208S001). The mice were maintained in a specific pathogen-free (SPF) environment, in accordance with the requirements of the Experimental Animal Ethics Committee of Xuzhou Medical University. They were subjected to a 12-hour light-dark cycle, provided ad libitum access to food and water, and allowed to acclimate for one week. All animal care and experimental procedures complied with the WMA Statement on animal use in biomedical research. Female BALB/c and NCG mice (6–8 weeks old; GemPharmatech) were intravenously injected with 2 × 10⁵ 4T1-luc cells (pretreated with or without 50 ng/mL IFN-γ for 24 h). Metastasis was monitored via bioluminescence imaging (IVIS Spectrum, PerkinElmer) after intraperitoneal injection of D-luciferin (150 mg/kg; Promega). Lungs were harvested on day 8, fixed in 4% PFA, and paraffin-embedded sections were stained with hematoxylin and eosin (H&E) for the quantification of metastatic nodules.

​​Immunoprecipitation and protein docking​​

Co-immunoprecipitation (Co-IP) was performed as previously described. Briefly, MDA-MB-231 lysates were incubated with anti-PD-L1 (CST, #13684) or anti-POLR2A (CST, #2629) antibodies overnight, followed by Protein A/G agarose beads (Beyotime, P2179M). Bound proteins were detected via Western blotting. Protein-protein interactions between PD-L1 and POLR2A were predicted using ZDOCK (Discovery Studio 2.25).

​​Bioinformatic analysis​​

ChIP-seq data for PD-L1 and POLR2A binding at the LY6E promoter region were analyzed using the Cistrome DB. TCGA-TNBC data were downloaded from UCSC Xena for correlation analysis of CD274 (encoding PD-L1) and LY6E expression using Pearson’s correlation analysis.

​​Statistical analysis

All data were analyzed using GraphPad Prism 9.4.0 software. Student’s t-test was used for two-group comparisons, and one-way ANOVA was used for multiple-group comparisons. Data are presented as mean ± standard deviation (SD).

​​Results​​

​ IFN-γ signaling enrichment in a metastatic TNBC subpopulation​​

To investigate the mechanisms underlying lung metastasis of TNBC cells, we performed an integrated reanalysis of single-cell RNA sequencing (scRNA-seq) data from TNBC lung metastases (PRJNA706068) retrieved from the Sequence Read Archive (SRA) database [28]. This original study involved transplanting TNBC patient-derived xenografts (PDXs) into murine mammary fat pads, followed by flow cytometry-based sorting of single tumor cells from the resulting lung metastases and primary orthotopic tumors (Fig. 1A). Principal component analysis (PCA) revealed indistinct cell clustering, indicating minimal global transcriptional differences between the majority of metastatic and primary tumor cells. Therefore, conventional differential gene expression analysis between the two groups proved insufficient to identify cell-level heterogeneity. Scrutiny of the PCA plot identified a subset of cells exhibiting significantly high PC1 values (PC1 >20), and corresponding differential gene expression heatmaps confirmed distinct transcriptional profiles in these cells (Fig. 1B-C). We designated cells with PC1 >20 as the “High_PC1” subpopulation and the remaining cells as the “Low_PC1” subpopulation (Fig. 1D). Subsequent analysis demonstrated significant enrichment of the IFN-γ signaling pathway within the High_PC1 subpopulation (Fig. 1E). Key pathway genes, including CD274, STAT1, and CXCL10, were upregulated compared to Low_PC1 cells and primary tumor cells; these genes are core enriched genes in the GSEA result of “INTERFERON_GAMMA_RESPONSE” (Fig. 1F, Fig S1). The pronounced enrichment of IFN-γ signaling specifically in a highly heterogeneous subpopulation of metastatic TNBC cells suggests a potentially significant role of this pathway in TNBC lung metastasis.

Fig. 1.

Fig. 1

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IFN-γ signaling is enriched in a distinct subpopulation of metastatic TNBC cells.​​ A​​ Schematic workflow for analyzing the mechanisms of TNBC lung metastasis using single-cell RNA sequencing data derived from primary orthotopic tumors and lung metastases in PDX models. Tumor cells were isolated via flow cytometry-based sorting. ​​B​​ PCA plot of all tumor cells. Cells from lung metastases (red) and primary sites (blue) show minimal global transcriptional separation. Cells derived from three distinct TNBC PDX sources (M1, M2, M3) are indicated. ​​C​​ Heatmap of differentially expressed genes (DEGs) between lung metastatic cells and primary tumor cells. Red indicates upregulation, blue indicates downregulation. Arrows highlight cells with aberrant expression profiles. ​​D​​ Schematic illustrating the stratification of cells into “High_PC1” (PC1 > 20) and “Low_PC1” (PC1 ≤ 20) subpopulations based on PCA results. ​​ E ​​ Significantly enriched KEGG pathways (FDR < 0.05) in the High_PC1 subpopulation identified by GSEA. ​​F​​ Expression levels (normalized counts) of key IFN-γ signaling pathway genes (CD274, STAT1, CXCL10) in High_PC1 versus Low_PC1 subpopulations and primary tumor cells. Data are presented as mean ± SD. Student’s t-test was used for two-group data analysis, while One Way ANOVA was used for multiple-group data analysis

IFN-γ activation promotes immune-independent TNBC metastasis

To elucidate the role of IFN-γ in lung metastasis of triple-negative breast cancer (TNBC), we treated the mouse TNBC cell lines 4T1 and EMT6 with IFN-γ, as referenced in prior studies, to mimic activation of the IFN-γ signaling pathway [9, 29]. Given the reported IFN-γ-induced apoptosis in tumor cells [30, 31], CCK-8 assays confirmed no significant suppression of 4T1 cell viability at concentrations up to 100 ng/mL, while EMT6 cells showed approximately 30% inhibition (Figs. 2A, S2A). Furthermore, treatment with 50 ng/mL IFN-γ sufficiently induced phosphorylation of the key downstream effectors JAK1 and STAT1 and significantly upregulated PD-L1 expression at the transcriptional level in both 4T1 and EMT6 cells (Figs. 2B–C, S2B–C). Comparable PD-L1 upregulation was observed at the protein level, with no significant difference between 50 ng/mL and 100 ng/mL IFN-γ treatments (Figs. 2D, S2D). Consequently, 4T1 cells pretreated with 50 ng/mL IFN-γ for 24 h were intravenously injected into both immunocompetent BALB/c mice and severe combined immunodeficient (SCID) NCG mice. IFN-γ-pretreated cells exhibited significantly enhanced lung metastatic nodule formation in both mouse models compared to untreated control cells (Fig. 2E–F). Histological analysis via H&E staining revealed a greater number of metastatic nodules with larger diameters in the lungs of mice injected with IFN-γ-pretreated cells (Fig. 2G–H). These results indicate that activation of the IFN-γ signaling pathway in 4T1 cells robustly promotes lung metastasis, and this effect was observed in both immunocompetent and severe combined immunodeficient hosts.

Fig. 2.

Fig. 2

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Activation of IFN-γ signaling promotes TNBC lung metastasis in an immune-independent manner.​​ A​​ Cell viability of 4T1 cells assessed by CCK-8 assay after 24 h treatment with indicated concentrations of IFN-γ. ​​B​​ Western blot analysis of total and phosphorylated STAT1 and JAK1 in 4T1 cells treated with 50 ng/mL IFN-γ for 24 h. ​​C​​ qPCR analysis of Cd274 (PD-L1) mRNA levels in 4T1 cells treated with indicated IFN-γ concentrations for 24 h. ​​D​​ Flow cytometry analysis of cell surface PD-L1 expression on 4T1 cells treated with 0-100 ng/mL IFN-γ for 24 h. Representative bioluminescence images (left) and quantification of total flux (right) of lungs from E​​ immunocompetent BALB/c mice (n = 7 per group) or (F)​​ severe combined immunodeficient mice NCG mice (n = 6 per group) 7 days after intravenous injection of 2 × 105 4T1-luc cells pre-treated with or without 50 ng/mL IFN-γ for 24 h. ​​G, H​​ Representative H&E-stained lung sections (left) and quantification of metastatic nodules (right) from (G)​​ BALB/c mice (n = 7 per group) and H​​ NCG mice (n = 5 per group) 7 days post-injection. Data are presented as mean ± SD. Student’s t-test was used for two-group data analysis, while One Way ANOVA was used for multiple-group data analysis

PD-L1 mediates IFN-γ-driven metastasis via immune-checkpoint-independent mechanism

To investigate whether PD-L1 contributes to IFN-γ-mediated lung metastasis of TNBC cells and to determine if this role is independent of its immune checkpoint function, we generated stable PD-L1 knockout (KO) 4T1 cells using CRISPR/Cas9 (Fig. 3A–C). We injected IFN-γ-pretreated PD-L1 wild-type (WT) or PD-L1 KO 4T1 cells into BALB/c and NCG mice to establish lung metastasis models. IFN-γ pretreatment enhanced lung metastatic nodule formation in PD-L1 WT cells but failed to do so in PD-L1 KO cells in immunocompetent BALB/c mice (Fig. 3D–E). Critically, this effect was consistent across both mouse models, as confirmed by in vivo imaging in NCG mice (Fig. 3H). Moreover, histopathological evaluation confirmed that PD-L1 knockout reversed the IFN-γ-induced increase in lung metastatic nodules, and this effect remained consistent across mice with distinct immune statuses (Fig. 3G–H).​ These findings demonstrate that PD-L1 is involved in the pro-metastatic effect of IFN-γ-mediated lung metastasis in TNBC cells.​

Fig. 3.

Fig. 3

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PD-L1 mediates IFN-γ-driven metastasis via an immune-independent mechanism.​​ A Schematic representation of PD-L1 genomic loci and CRISPR/Cas9-targeted sites in generated 4T1 PD-L1 knockout cell lines. B Western blot analysis of PD-L1 protein expression in PD-L1 wild-type (WT) and KO 4T1 cells treated with 0–50 ng/mL mouse IFN-γ for 24 h. C Flow cytometry analysis confirming surface PD-L1 expression on PD-L1 WT and KO 4T1 cells treatment with or without 50 ng/mL IFN-γ for 24 h. D Representative bioluminescence images of BALB/c mice (n = 7 per group) 7 days after intravenous injection of PD-L1 WT or KO 4T1-luc cells pre-treated with or without 50 ng/mL IFN-γ. E Quantification of total bioluminescent flux from D. F Representative bioluminescence images and quantification of total bioluminescent flux from NCG mice (PD-L1 WT Control n = 3, other groups n = 6) injected with indicated cells. Representative H&E-stained lung sections (left) and quantification of metastatic nodules (right) from (G)​​ BALB/c mice (n = 7 per group) and (H)​​ NCG mice (PD-L1 WT Control n = 3, other groups n = 5), 7 days post-injection. Data are presented as mean ± SD. Student’s t-test was used for two-group data analysis, while One Way ANOVA was used for multiple-group data analysis

PD-L1 loss impairs metastasis-specific functions without affecting proliferation

To establish the role of PD-L1 in 4T1 cell lung metastasis and determine whether the PD-L1 knockout-mediated reduction in lung metastasis involves impaired proliferation, we first performed intravenous injection of PD-L1 KO 4T1 cells into both immunocompetent BALB/c mice and severe combined immunodeficient NCG mice. This significantly suppressed lung metastasis formation in both models, suggesting a non-canonical, immune checkpoint-independent pro-metastatic function of PD-L1 (Fig. 4A–B). Second, functional in vitro assays performed in the absence of an immune microenvironment revealed that PD-L1 knockout significantly impaired cell migration and invasion (Fig. 4C–D), while cell proliferation and clonogenicity remained unaffected (Fig. 4E–F). Furthermore, PD-L1 knockout did not affect subcutaneous tumor growth in vivo (Fig. 4G), indicating that the observed reduction in metastasis was not attributable to impaired proliferation. Since the immune checkpoint mechanism of PD-L1 primarily relies on T cells, and NCG mice are devoid of T cells, B cells, and NK cells, exhibit deficient complement activity, and have functional deficiencies in dendritic cells and macrophages—coupled with the immune microenvironment-free conditions of in vitro migration assays—these findings collectively indicate that PD-L1 contributes to IFN-γ-mediated lung metastasis in TNBC cells through a non-canonical, immune checkpoint-independent mechanism.

Fig. 4.

Fig. 4

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PD-L1 Loss impairs metastasis-specific functions without affecting proliferation.​​ Representative bioluminescence images (left) and quantification of total flux (right) of lungs from A​​ immunocompetent BALB/c mice (n = 5 per group) or B​​ severe combined immunodeficient mice NCG mice (n = 9 per group), 7 days after intravenous injection of 4T1-WT and 4T1 PD-L1 KO cells. C Representative images and D quantification of migration (24 h) and invasion (48 h) of PD-L1 WT and KO 4T1 cells using Transwell assays (n = 6 wells per group). E Cell proliferation of PD-L1 WT and KO 4T1 cells assessed by CCK-8 assay over 72 h. F Representative images (left) and quantification (right) of colony formation by PD-L1 WT and KO 4T1 cells (n = 3 wells per group). G Tumor growth curves (left), representative endpoint tumor images (middle), and tumor weights (right; n = 5 mice per group) of PD-L1 WT and KO 4T1 cells subcutaneously implanted in NCG mice. Data are presented as mean ± SD. Student’s t-test was used for two-group data analysis, while One Way ANOVA was used for multiple-group data analysis

​IFN-γ promotes PD-L1 nuclear translocation via HDAC2-mediated deacetylation​

To elucidate how IFN-γ facilitates PD-L1-mediated TNBC lung metastasis, we treated the human TNBC cell line MDA-MB-231 with varying concentrations of IFN-γ. IFN-γ treatment increased both total PD-L1 expression (Fig. 5A) and nuclear PD-L1 levels (Fig. 5B). ​​Immunofluorescence staining confirmed that IFN-γ treatment promoted PD-L1 translocation to the nucleus in MDA-MB-231 cells, resulting in increased colocalization with the nuclear marker DAPI (Fig. 5C). Currently reported pathways for PD-L1 nuclear import involve HDAC2-mediated deacetylation and phosphorylated STAT3 (p-STAT3) signaling [13, 14]. In our study, IFN-γ stimulation did not increase nuclear p-STAT3 levels in MDA-MB-231 cells (Fig. 5D). Furthermore, pretreatment with the p-STAT3 inhibitor S3I-201 failed to suppress IFN-γ-induced nuclear PD-L1 accumulation (Fig. 5E), indicating that IFN-γ does not use the p-STAT3 pathway to promote PD-L1 nuclear translocation. Conversely, IFN-γ treatment significantly reduced PD-L1 acetylation (Fig. 5F), suggesting that it promotes PD-L1 deacetylation. Inhibition of the HDAC2-mediated deacetylation pathway using two distinct inhibitors effectively blocked IFN-γ-induced upregulation of nuclear PD-L1 (Fig. 5G). Critically, in the in vivo lung metastasis model, the HDAC2 inhibitor Santacruzamate A significantly attenuated the pro-metastatic effect of IFN-γ (Fig. 5H). These collective findings indicate that IFN-γ likely promotes nuclear PD-L1 translocation and TNBC lung metastasis via the HDAC2 deacetylation pathway.​​

Fig. 5.

Fig. 5

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IFN-γ promotes PD-L1 nuclear translocation via HDAC2-mediated deacetylation.​​ Western blot analysis of A​​ total PD-L1 protein levels and B nuclear PD-L1 in MDA-MB-231 cells treated with 0–50 ng/mL human IFN-γ for 48 h. ​​C Representative Immunofluorescence staining images assessed the subcellular localization of PD-L1 in IFN-γ-stimulated MDA-MB-231 cells (left), colocalization analysis of PD-L1 and DAPI by ImageJ software shown in the right panel.​ D Western blot analysis of cytoplasmic and nuclear fractions from MDA-MB-231 cells treated with 50 ng/mL IFN-γ for 48 h, probed for STAT3 and phosphorylated STAT3. ​​E​​ Western blot analysis of cytoplasmic and nuclear PD-L1 in MDA-MB-231 cells treated with 100 µM STAT3 inhibitor S3I-201 and 50 ng/mL IFN-γ for 48 h. ​​F​​ Immunoprecipitation (IP) of PD-L1 followed by Western blot for acetylated lysine (Ac-K) in MDA-MB-231 cells treated with or without 50 ng/mL IFN-γ for 48 h. ​​G​​ Western blot analysis of nuclear PD-L1 levels in MDA-MB-231 cells treated with 20 µM HDAC2 inhibitor Santacruzamate A or 25 µM Importin α/β inhibitor Ivermectin followed by 50 ng/mL IFN-γ for 48 h. ​​H​​ Representative images of lungs and quantification of metastatic nodules from BALB/c mice (n = 6 per group) fixed in Bouin’s solution 10 days after intravenous injection of 4T1 cells pre-treated with Santacruzamate A for 24 h, followed by IFN-γ treatment for 24 h. Data are presented as mean ± SD. Student’s t-test was used for two-group data analysis, while One Way ANOVA was used for multiple-group data analysis

LY6E identified as key downstream effector of nuclear PD-L1 in metastasis​

To identify key downstream effectors of nuclear PD-L1 in IFN-γ-promoted TNBC lung metastasis, we performed RNA sequencing (RNA-seq) on PD-L1 wild-type (WT) and PD-L1 knockout (KO) 4T1 cells treated with or without IFN-γ. Differentially expressed genes (DEGs) were filtered using four stringent criteria: (i) responsiveness to IFN-γ stimulation; (ii) dependence on PD-L1 expression; (iii) loss of responsiveness to IFN-γ in PD-L1 KO cells; and (iv) opposite regulatory trends following IFN-γ stimulation versus PD-L1 knockout alone. This analysis identified four candidate genes that met al.l the criteria (Fig. 6A). Subsequent qPCR validation confirmed Cldn1 and Ly6e as the most significantly differentially expressed genes (Fig. 6B–C), with changes in protein expression mirroring those in transcriptional levels (Figs. 6D, S3A–B). Using the human TNBC cell line MDA-MB-231, we further assessed transcriptomic changes in LY6E and CLDN1 following PD-L1 knockdown. LY6E exhibited consistent transcriptional changes with the murine 4T1 cell model (Fig. 6E), whereas CLDN1 showed no significant increase following IFN-γ stimulation (Fig. 6F). Subsequent validation at the protein level confirmed interspecies consistency for both targets. IFN-γ significantly upregulated LY6E expression, an effect reversed by PD-L1 knockdown (Fig. 6G). In contrast, CLDN1 displayed inconsistent changes across species (Fig. 6H).​ Clinically, both PD-L1 (encoded by CD274) and LY6E mRNA levels were concomitantly upregulated in TNBC patients’ tumors compared to adjacent normal tissues (Fig. 6I) and showed a significant positive correlation (Fig. 6J). In addition, the HDAC2 inhibitor Santacruzamate A significantly inhibited the IFN-γ-induced increase in Ly6e expression (Fig S3C). LY6E exhibits low expression in normal tissues but high expression in multiple malignancies, positioning it as a potential therapeutic target across solid tumors [32, 33]. Omics studies indicate that LY6E expression levels predict lung metastatic potential of breast cancer. Elevated LY6E expression is significantly correlated with poor prognosis in breast cancer patients [24]. Furthermore, LY6E is essential for TGF-β signal transduction, contributing to the phosphorylation of Smad1/5 and Smad3, and promotes drug resistance and immune evasion in breast cancer cells [25].​ ​​ The transcription factor GATA3 has been reported to suppress breast cancer lung metastasis, where its overexpression inhibits LY6E expression, while its knockdown conversely upregulates LY6E expression [26].​ ​​Consistent with previous reports, our study demonstrates that LY6E overexpression promotes pulmonary metastasis in TNBC cells (Fig. 6K).​ Collectively, these findings identify LY6E as a key downstream target mediating the pro-metastatic effects of IFN-γ-driven nuclear PD-L1 in TNBC.

Fig. 6.

Fig. 6

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LY6E is identified as a key downstream effector of nuclear PD-L1 in metastasis.​​ A​​ Left: Venn diagram illustrating the strategy for identifying downstream targets of IFN-γ/nuclear PD-L1 from RNA-seq data of PD-L1 WT and KO 4T1 cells treated with or without 50 ng/mL IFN-γ for 24 h. Right: Heatmap of four candidate genes. ​​B–C​​ qPCR validation of Cldn1, Ly6e, Anxa8, and Gnb4 mRNA expression in the indicated treatment groups of 4T1 cells (n = 4). ​​D​​ Western blot analysis of Claudin-1 and Ly6E protein expression in PD-L1 WT and KO 4T1 cells treated with or without 50 ng/mL IFN-γ for 24 h. ​​E–F​​ qPCR analysis of CLDN1 and LY6E mRNA expression in PD-L1 WT and PD-L1 KO MDA-MB-231 cells treated with or without 50 ng/mL IFN-γ for 48 h. ​​G–H​​ Western blot analysis of Claudin-1 and Ly6E protein expression in PD-L1 WT and KO MDA-MB-231 cells treated with or without 50 ng/mL IFN-γ for 48 h. I​​ Analysis of LY6E and CD274 mRNA expression in TCGA TNBC tumor and adjacent normal tissue samples. ​​J​​ Correlation analysis of LY6E and CD274 mRNA expression in TCGA TNBC samples. ​​K​​ Representative images of lungs and quantification of metastatic nodules from LY6E overexpression cells. Data are presented as mean ± SD. Student’s t-test was used for two-group data analysis, while One Way ANOVA was used for multiple-group data analysis

​Nuclear PD-L1 forms transcriptional complex with POLR2A to upregulate LY6E​

To elucidate the mechanism by which nuclear PD-L1 upregulates LY6E, we analyzed published PD-L1 chromatin immunoprecipitation sequencing (ChIP-seq) data in MDA-MB-231 cells. This analysis revealed no significant enrichment of PD-L1 binding peaks within the LY6E promoter region (Fig. 7A), excluding direct transcriptional activation of LY6E by nuclear PD-L1. Given prior reports that PD-L1 can form complexes with transcription factors (TFs) to activate target genes, we hypothesized a similar indirect mechanism for LY6E regulation. Mining ChIP-seq data from the Cistrome DB [34] for MDA-MB-231 cells identified seven proteins with binding peaks at the LY6E promoter and positive transcriptional potential scores (Fig. 7B). Cross-referencing with published PD-L1 interactome data [14] (Co-IP/MS in MDA-MB-231 cells; 1297 binding partners, including 104 TFs) revealed POLR2A as the sole protein meeting two criteria: (i) direct binding to PD-L1 and (ii) significant enrichment at the LY6E promoter (Fig. 7C). POLR2A binding at the LY6E promoter region was further confirmed in MDA-MB-231 cells [35, 36] (Fig. 7D). Protein-protein docking using the ZDOCK module of Discovery Studio predicted strong interactions between PD-L1 and POLR2A, specifically a salt bridge between PD-L1 R140 and POLR2A E517, electrostatic interactions involving PD-L1 K136 and K185, and POLR2A D452 and D663, as well as multiple hydrogen bonds (< 2.5 Å) (Fig. 7E). Critically, co-immunoprecipitation (Co-IP) confirmed an IFN-γ-dependent physical interaction between PD-L1 and POLR2A (Fig. 7F). Collectively, ChIP-seq analysis, Co-IP/MS data, protein docking, and experimental validation support a model wherein PD-L1 binds POLR2A to form a transcriptional complex that activates LY6E expression.

Fig. 7.

Fig. 7

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Nuclear PD-L1 forms a transcriptional complex with POLR2A to upregulate LY6E.​​ A​​ Browser view of published ChIP-seq data showing lack of PD-L1 enrichment at the LY6E promoter region (highlighted in yellow) in MDA-MB-231 cells. ​​B​​ Transcription factors (TFs) predicted to bind the LY6E promoter in MDA-MB-231 cells, ranked by transcriptional potential score. ​​C​​ Venn diagram identifying POLR2A as the only TF overlapping between factors predicted to bind LY6E (B) and factors found to interact with PD-L1 by Co-IP/MS in MDA-MB-231 cells. ​​D​​ Browser view of ChIP-seq data showing POLR2A enrichment at the LY6E promoter region (highlighted in yellow) in MDA-MB-231 cells. ​​E​​ Predicted structural model of the PD-L1/POLR2A interaction generated by ZDOCK. Key interacting residues are labeled: Salt bridge (PD-L1 R140 - POLR2A E517), Electrostatic interactions (PD-L1 K136/K185 - POLR2A D452/D663). ​​F​​ Co-immunoprecipitation assay in MDA-MB-231 cells to detect protein interaction between POLR2A and PD-L1

​​Discussion

Our study uncovers a non-canonical metastasis-driving axis in TNBC wherein IFN-γ promotes the nuclear translocation of PD-L1, which subsequently interacts with POLR2A to transcriptionally activate the pro-metastatic factor LY6E. This mechanism provides a compelling explanation for the clinical paradox wherein PD-L1-high TNBC patients exhibit poor responses to anti-PD-L1 immunotherapy yet develop aggressive lung metastases [3–5]. Critically, our work demonstrates that PD-L1’s metastatic function operates independently of its immune checkpoint role, as evidenced by persistent promotion of metastasis in immunodeficient models upon IFN-γ stimulation (Figs. 2 and 3).

The identification of nuclear PD-L1 as a critical metastasis driver fundamentally expands our understanding of PD-L1 biology beyond its canonical immune checkpoint function. While prior studies have established that nuclear PD-L1 regulates the DNA damage response in TNBC [11] and modulates chromatin dynamics in other cancers [12, 14], our work is the first to demonstrate its direct involvement in transcriptional reprogramming of metastasis-associated genes. This nuclear function persists even in severe combined immunodeficient (SCID) mouse models, confirming its independence from T cell-mediated immunity. Importantly, the HDAC2-dependent nuclear translocation pathway we identified (Fig. 5) is consistent with Gao et al.’s report [14] that PD-L1 acetylation status dictates its subcellular localization, suggesting that HDAC inhibitors may suppress metastasis by blocking PD-L1 nuclear trafficking—though their lack of specificity remains a therapeutic challenge.

LY6E emerges as a key effector that linking nuclear PD-L1 to metastasis. Our data corroborate earlier reports of LY6E’s involvement in breast cancer progression [24] while providing mechanistic insight into its transcriptional induction via the PD-L1-POLR2A complex (Figs. 6 and 7). The clinical correlation between PD-L1 and LY6E in TNBC specimens reinforces their pathological significance. Mechanistically, LY6E’s known potentiation of TGF-β/Smad signaling offers a plausible explanation for its pro-metastatic function, as TGF-β is a master regulator of metastatic niche formation [25].

The discovery that PD-L1 interacts with POLR2A to form a transcription factor complex represents a paradigm shift in understanding PD-L1’s nuclear functions. While we provide biochemical evidence for PD-L1-POLR2A interaction through structural modeling (Fig. 7E) and co-immunoprecipitation (Fig. 7F), our study has not functionally validated the requirement of this interaction for LY6E transactivation. Crucially, we have not demonstrated that disrupting PD-L1-POLR2A binding selectively inhibits PD-L1’s transcriptional activity without globally compromising POLR2A function-a significant knowledge gap given POLR2A’s essential role in basal transcription [35, 36]. Future studies employing POLR2A point mutants deficient in PD-L1 binding or small-molecule disruptors are needed to establish causality. This represents both a limitation and an opportunity, as selectively targeting this interface may mitigate toxicity concerns associated with direct POLR2A inhibition.

Therapeutically, our findings rationalize dual targeting of PD-L1’s membranous and nuclear functions. The inability of anti-PD-L1 antibodies to neutralize nuclear PD-L1 likely contributes to treatment failure in metastatic TNBC. Promisingly, virtual screening approaches may identify compounds that disrupt the PD-L1-POLR2A interaction interface. Such agents might synergize with existing immunotherapies by concurrently blocking PD-1/PD-L1 interactions and suppressing nuclear PD-L1-driven transcriptional programs. Although our study establishes the PD-L1-POLR2A-LY6E axis in vitro and in murine models, validation in patient-derived organoids or human metastatic biopsies would strengthen clinical relevance. Additionally, the contribution of tumor microenvironmental factors such as myeloid-derived suppressor cells to this pathway warrants exploration, as IFN-γ can originate from both T cells and innate immune populations during immunotherapy [37–39].

Conclusion

In summary, we delineate a previously unrecognized IFN-γ- nuclear PD-L1-POLR2A-LY6E pathway that drives lung metastasis in TNBC independently of immune checkpoint mechanisms (Fig. 8). This work not only explains the limited efficacy of anti-PD-L1 therapy in metastasis prevention but also provides a mechanistic foundation for novel therapeutic combinations. Targeting nuclear PD-L1 complexes or their downstream effectors like LY6E represents a promising strategy to reduce metastatic recurrence in TNBC patients.

Fig. 8.

Fig. 8

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Mechanism of lung metastasis progression in triple-negative breast cancer patients despite anti-PD-L1 antibody therapy​

Supplementary Information

Below is the link to the electronic supplementary material

Supplementary Material 1 (467.5KB, docx)
Supplementary Material 2 (1.8MB, docx)
Supplementary Material 3 (5.4MB, xls)

Acknowledgements

We would like to thank EditChecks (https://editchecks.com.cn/) for providing linguistic assistance during the preparation of this manuscript.

Abbreviations

TNBC

Triple-negative breast cancer

PD-L1

Programmed death-ligand 1

IFN-γ

Interferon-gamma

LY6E

Lymphocyte antigen 6E

POLR2A

RNA polymerase II subunit A

PCA

Principal component analysis

GSEA

Gene set enrichment analysis

PDX

Patient-derived xenografts

CCK-8

Cell counting Kit-8

H&E

Hematoxylin and eosin

HDAC2

Histone deacetylase 2

ChIP-seq

Chromatin immunoprecipitation sequencing

Co-IP/MS

Co-immunoprecipitation/mass spectrometry

RNA-seq

RNA sequencing

TCGA

The cancer genome atlas

Author contributions

Xu Wang: Data curation, Funding acquisition, Investigation, Methodology, Formal analysis, Writing—review and editing. Qi Zhou: Data curation, Investigation, Formal analysis, Methodology, Writing—original draft. Pu Wang: Methodology, Investigation. Shunshun Bao: Data curation. Xianzheng Wei: Methodology. Rui Hou: Formal analysis. Xuan Zhao: Methodology. Sijin Li: Methodology. Zhangchun Guan: Formal analysis.: Data curation. Wen Ma: Methodology. Junnian Zheng: Funding acquisition, Project administration, Writing—review. Dan Liu and Ming Shi: Conceptualization, Funding acquisition, Project administration, Investigation, Methodology, Writing—review.

Funding

This work was supported by the National Natural Science Foundation of China (No.: 82471378, 82304535, 81972719, 82273207, and 82003164), Natural Science Foundation of Jiangsu Province (No.: BK20220671, BK20210910, BK20201012, and BK20210913), National science research in Universities of Jiangsu Province (No.: 22KJB320009, 21KJA320008 and 21KJB320021), Outstanding Talents Research Startup Fund of Xuzhou Medical University (D2021049). Jiangsu Provincial Young Science and Technology Talent Lifting Program (JSTJ-2025-496).

Data availability

The RNA-seq transcriptomic data used in this article can be downloaded from the Zenodo database. Download link: https://doi.org/10.5281/zenodo.17471657. Single-cell sequencing data for orthotopic and lung metastases of triple-negative breast cancer can be downloaded from the National Center for Biotechnology Information Sequence Read Archive (SRA) database with the accession number PRJNA706068.

Declarations

Ethics approval and consent to participate

The animal studies were reviewed and approved by the Animal Ethics Committee of Xuzhou Medical University. Animal experiment approval number: 202208S001. Registry and the Registration No. of the Study/Trial: Not applicable.

Consent for publication

Not applicable.

Competing interests

The authors declare no competing interests.

Footnotes

Publisher’s note

Springer Nature remains neutral with regard to jurisdictional claims in published maps and institutional affiliations.

Xu Wang, Qi Zhou and Pu Wang have contributed equally to this work.

Contributor Information

Junnian Zheng, Email: jnzheng@xzhmu.edu.cn.

Dan Liu, Email: liudan_bd@sina.com.

Ming Shi, Email: mingshi@xzhmu.edu.cn.

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Associated Data

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

Supplementary Materials

Supplementary Material 1 (467.5KB, docx)
Supplementary Material 2 (1.8MB, docx)
Supplementary Material 3 (5.4MB, xls)

Data Availability Statement

The RNA-seq transcriptomic data used in this article can be downloaded from the Zenodo database. Download link: https://doi.org/10.5281/zenodo.17471657. Single-cell sequencing data for orthotopic and lung metastases of triple-negative breast cancer can be downloaded from the National Center for Biotechnology Information Sequence Read Archive (SRA) database with the accession number PRJNA706068.

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