Abstract

Background & Aims

   The programmed death-ligand 1 (PD-L1) is a major co-inhibitory
   checkpoint factor that controls T-cell activities in tumours. PD-L1 is
   expressed on immune cells and tumour cells. Whether tumour
   cell-expressed PD-L1 affects tumour cells in an immune cell-independent
   fashion remains largely elusive. In this study, we investigated the
   significance of tumour cell-expressed PD-L1 with a focus on downstream
   signals and changes in lipid metabolism.

Methods

   Immune-independent functions of PD-L1 in tumour growth were
   investigated in vitro and in immuno-deficient mice in vivo. The global
   influence of PD-L1 in targeted/untargeted lipidomic metabolites was
   studied by comprehensive mass spectrometry-based metabolomic analysis
   in liver cancer. Effects on lipid metabolism were confirmed by
   triglyceride and cholesterol assays as well as by Oil Red O staining in
   liver, pancreatic, breast, and oesophageal squamous cancer. Underlying
   mechanisms were investigated by real-time quantitative PCR, Western
   blot analysis, co-immunoprecipitation, pull-down assays,
   immunofluorescence staining, and RNA sequencing.

Results

   PD-L1 enhanced the accumulation of triglycerides, cholesterol, and
   lipid droplets in tumours. PD-L1 influenced targeted/untargeted
   lipidomic metabolites in hepatoma, including lipid metabolism, glucose
   metabolism, amino acid metabolism, nucleotide metabolism, and energy
   metabolism, suggesting that PD-L1 globally modulates the metabolic
   reprogramming of tumours. Mechanistically, PD-L1 activated epidermal
   growth factor receptor (EGFR) and/or integrin β4 (ITGB4) by forming a
   complex of PD-L1/EGFR/ITGB4 in the cell membrane, prior to activating
   PI3K/mTOR/SREBP1c signalling, leading to reprogramming of lipid
   metabolism in tumours. Functionally, PD-L1-mediated lipid metabolism
   reprogramming supported the tumour growth in vitro and in vivo through
   EGFR and/or ITGB4 in an immune cell-independent manner.

Conclusions

   Our findings on lipogenesis and EGFR activation by tumour
   cell-expressed PD-L1 suggest that, in addition to its immunostimulatory
   effects, anti-PD-L1 may restrict lipid metabolism and EGFR/ITGB4
   signalling in liver cancer therapy.

Impact and implications

   In this study, we present evidence that PD-L1 drives the reprogramming
   of lipid metabolism in tumours. PD-L1 forms a complex with epidermal
   growth factor receptor (EGFR) and ITGB4, activating the
   PI3K/Akt/mTOR/SREBP1c signalling pathway and thereby contributing to
   lipid metabolism in cancer progression. Our findings offer novel
   insights into the mechanisms by which PD-L1 initiates the reprogramming
   of lipid metabolism in tumours. From a clinical perspective, the
   anti-PD-L1 antibody may alleviate resistance to the anti-EGFR antibody
   cetuximab and inhibit the reprogramming of lipid metabolism in tumours.

   Keywords: PD-L1, Tumours, Lipid metabolism reprogramming, EGFR, ITGB4,
   SREBP1c

Graphical abstract

   [53]graphic file with name ga1.jpg
   [54]Open in a new tab

Highlights

     * •
       Tumour cell-expressed PD-L1 drives reprogramming of lipid
       metabolism in tumours.
     * •
       PD-L1 forms a complex with EGFR/ITGB4 to trigger reprogramming of
       lipid metabolism in HCC.
     * •
       PD-L1 triggers reprogramming of lipid metabolism via
       EGFR/ITGB4/PI3K/Akt/mTOR/SREBP1c/FASN/ACLY signalling in tumours.
     * •
       PD-L1-mediated reprogramming of lipid metabolism supports tumour
       growth in vitro and in vivo.

Introduction

   The programmed death-ligand 1 (PD-L1), encoded by CD274, serves as the
   programmed cell death-1 (PD-1) ligand, a key co-inhibitory checkpoint
   signalling molecule regulating T-cell activities. Various cancers
   exhibit elevated PD-L1 levels, correlating with a dismal prognosis, and
   exploit the PD-L1/PD-1 signalling pathway to evade T-cell
   immunity.[55]^1 Elevated lipid biosynthesis not only enhances the
   immunosuppressive activity of regulatory T cells (Tregs) but also
   facilitates glucose metabolism in cervical cancer mediated by
   PD-L1.[56]^2 However, the relationship between tumour cell-expressed
   PD-L1 and lipid metabolism reprogramming in cancers is poorly
   understood.

   Reprogramming of lipid metabolism assumes a pivotal role in tumour
   development.[57]^3^,[58]^4 Critical lipogenic enzymes, including
   stearoyl-CoA desaturase (SCD), fatty acid (FA) synthase (FASN), and
   acetyl-CoA carboxylase,[59]^5 are implicated in hepatocarcinogenesis.
   In addition, sterol regulatory element-binding protein 1 (SREBP-1), a
   crucial transcription factor, upregulates ATP-citrate lyase (ACLY),
   FASN, and SCD, thereby promoting FA synthesis and augmenting
   cholesterol uptake in hepatocytes.[60]^6 Our previous research revealed
   that SPIN1 coactivates sterol regulatory element-binding protein-1c
   (SREBP1c) in the FASN promoter, contributing to the regulation of
   abnormal lipid metabolism.[61]^7 Furthermore, our group reported that
   cholesterol upregulates HULC in hepatoma cells.[62]^8 Although
   metabolomic studies have successfully integrated genomics and
   transcriptomics, offering deeper insights into disease mechanisms, the
   precise mechanism governing lipid metabolism reprogramming in tumours
   remains elusive.

   The epidermal growth factor receptor (EGFR) is a 170-kDa transmembrane
   glycoprotein cell surface receptor belonging to the ErbB/HER family.
   Activated EGFR influences four major signalling pathways: MAPK,
   phosphoinositide 3-kinase (PI3K)/Akt/mammalian/mechanistic target of
   rapamycin (mTOR), PLCγ/PKC, and JAK/STAT.[63]^9 Cetuximab, a
   human–mouse chimeric IgG1 monoclonal antibody, binds to domain III of
   the extracellular segment of the tethered inactive state of EGFR,
   directly blocking ligand binding.[64]^10 PD-L1 binding and its
   involvement in EGFR activation antagonise TRAIL-induced apoptosis in
   gastric cancer cells.[65]^11 The EGFR pathway regulates PD-L1
   expression via the IL-6/JAK/STAT3 signalling pathway in EGFR-mutated
   non-small cell lung cancer.[66]^12 However, whether EGFR-mediated lipid
   metabolism reprogramming is associated with PD-L1 expression in tumours
   remains unknown. Integrins, which are heterodimeric transmembrane
   receptors, mediate interactions between cells and extracellular matrix
   components.[67]^13 Integrin β4 (ITGB4) is aberrantly expressed in
   several cancers, including breast, colorectal, and lung, and is
   positively associated with poor prognosis.[68][14], [69][15], [70][16]
   ITGB4 regulates multiple signalling pathways, including ErbB2, PI3K,
   FAK/Akt, and c-Me, to promote tumour progression.[71][17], [72][18],
   [73][19], [74][20] However, the role of ITGB4 in lipid metabolism
   reprogramming in cancer remains unclear. The atypical serine/threonine
   kinase mTOR serves as the master modulator of cell metabolism and
   growth by inhibiting catabolic processes such as autophagy and
   promoting anabolic processes such as lipid, protein, nucleotide, FA
   synthesis, and ribosome biogenesis.[75]^21 mTOR signalling is typically
   triggered in cancer cells and promotes lipid metabolism by regulating
   the expression and/or activity of several key metabolic enzymes.[76]^22
   However, the effect of PD-L1 on mTOR signalling in the regulation of
   lipid metabolism is not well documented.

   In this study, we investigated immune-independent functions of PD-L1 in
   tumour cells, focusing on the modulation of lipid metabolism
   reprogramming. Strikingly, we found that PD-L1 triggered the lipid
   metabolism reprogramming in liver and gastrointestinal cancers.

Materials and methods

Cell lines and cell culture conditions

   The human immortalised normal liver LO2 cell line, Chang Liver cell
   line, pancreatic cancer cell line ASPC1, breast cancer cell line BT474,
   and oesophageal squamous cell carcinoma cell lines KYSE180 and KYSE2
   were cultured in RPMI-1640 medium (Gibco, Grand Island, NY, USA). Human
   hepatoma cell lines Huh7 and HepG2, pancreatic cancer cell line PANC1,
   and breast cancer cell line MCF7 were maintained in DMEM (Gibco). All
   cell lines were supplemented with heat-inactivated 10% FBS (Gibco),
   100 U/ml penicillin, and 100 μg/ml streptomycin; grown at 37 °C in a 5%
   CO[2] atmosphere; and routinely tested for mycoplasma. The cell lines
   were always mycoplasma negative. The cells were cultured in different
   flasks or plates for 36 or 48 h and then transfected with plasmids or
   siRNAs. Transfection was performed using the Lipofectamine 2000 reagent
   (Invitrogen, Carlsbad, CA, USA) according to the manufacturer’s
   protocol.

Statistical analysis

   Each experiment was repeated at least thrice. Statistical significance
   was assessed by comparing mean values (±SD) using Student’s t test for
   independent groups, with significance assumed for ∗p <0.05; ∗∗p <0.01;
   and ∗∗∗p <0.001.

Ethics approval and consent to participate

   All experiments involving human participants were performed in
   accordance with the Code of Ethics of the World Medical Association
   (Declaration of Helsinki). With informed consent from all participants,
   paired specimens of HCC and adjacent normal tissues were collected. All
   experiments involving human participants and animals were approved by
   the Institute Research Ethics Committee of Tianjin Medical University
   Cancer Institute and Hospital.

Results

PD-L1 triggers the accumulation of triglycerides, cholesterols, and lipid
droplets in tumours

   To understand the immune-independent effects of PD-L1, including the
   effects on lipid metabolites, we investigated lipid alterations in
   tumour and their correction with PD-L1 expression in hepatocellular
   carcinoma (HCC). Triglycerides (TGs) and cholesterols displayed
   remarkable accumulation in HCC tissues relative to their corresponding
   peritumoral tissues ([77]Fig. S1-1A and B). Intriguingly, a positive
   correlation existed between PD-L1 and TG and cholesterol levels in the
   clinical samples ([78]Fig. 1A and B). PD-L1 overexpression increased TG
   and cholesterol levels in tumour cells in a dose-dependent manner
   ([79]Fig. 1C and D, and [80]Fig. S1-1C–F). Conversely, PD-L1 knockdown
   reduced TG and cholesterol levels in these cells in a dose-dependent
   manner ([81]Fig. 1E and F, [82]Fig. S1-1G–L, and [83]Fig. S1-2A–C). Oil
   Red O staining revealed that PD-L1 overexpression strongly accelerated
   lipogenesis in KYSE180 cells ([84]Fig. 1G), whereas treatment with
   PD-L1 siRNA blocked this effect ([85]Fig. 1G and [86]Fig. S1-2D). In
   addition, PD-L1 knockout (KO) significantly decreased TG and
   cholesterol levels, whereas reconstitution of PD-L1 reversed this
   phenotype ([87]Fig. 1H and I, and [88]Fig. S1-3A).

Fig. 1.

   [89]Fig. 1
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   PD-L1 triggers the accumulation of triglycerides, cholesterols, and
   lipid droplets in tumours.

   (A and B) Investigation of the relationship between PD-L1 and
   triglycerides/cholesterols in 35 HCC tissues using RT-qPCR and tissue
   total triglyceride and cholesterol assay kits. (C and D) Assessment of
   the impact of PD-L1 overexpression on intracellular levels of
   triglycerides and cholesterols in tumour cells using tissue total
   triglyceride and cholesterol assay kits. (E and F) Determination of the
   effect of PD-L1 siRNA on intracellular triglycerides and cholesterols
   in tumour cells using tissue total triglyceride and cholesterol assay
   kits. (G) Evaluation of the influence of PD-L1 on lipid droplets in
   KYSE180 cells using Oil Red O staining. (H and I) Measurement of
   triglyceride and cholesterol levels after the overexpression of PD-L1
   plasmid in PD-L1 KO HepG2 cells. HCC, hepatocellular carcinoma; KO,
   knockout; PD-L1, programmed death-ligand 1; RT-qPCR, real-time
   quantitative PCR.

   We screened for the effect of PD-L1 on HCC gene expression using
   RNA-sequencing (RNA-Seq) analysis. The profiling data revealed that the
   expressions of 332 genes were upregulated, and those of 1,113 genes
   were downregulated by 1.5-fold when PD-L1 was knocked down in HepG2
   cells ([91]Fig. S1-3B). Interestingly, KEGG pathway analysis
   demonstrated that PD-L1 is involved in the biosynthesis of unsaturated
   FAs, the peroxisome proliferator-activated receptor (PPAR) signalling
   pathway, and cholesterol metabolism ([92]Fig. S1-3C). We validated the
   expression levels of PPAR signalling pathway-responsive genes using
   real-time quantitative PCR (RT-qPCR) in HepG2 cells ([93]Fig. S1-3D).

   The relapse-free survival analysis of patients with HCC revealed that
   those with high PD-L1 expression had lower survival rates in The Cancer
   Genome Atlas (TCGA) database ([94]Fig. S1-4A). Immunohistochemical
   (IHC) analysis showed that the PD-L1 positivity rate was 80% (64/80) in
   the HCC tissue microarray ([95]Fig. S1-4B). Moreover, PD-L1 levels
   significantly increased in 35 clinical HCC tissues relative to their
   corresponding peritumoral tissues ([96]Fig. S1-4C and D). Consistently,
   high PD-L1 levels were observed in hepatoma cell lines compared with
   normal liver cell lines ([97]Fig. S1-4E). Thus, we conclude that PD-L1
   accumulates TGs, cholesterols, and lipid droplets in tumours.

PD-L1 confers lipid metabolism reprogramming in liver cancer

   To further substantiate the influence of PD-L1 on lipid metabolism
   reprogramming, we conducted untargeted/targeted metabolite analysis
   using comprehensive mass spectrometry metabolomics. In the untargeted
   metabolite analysis, diverse metabolites were visually presented in the
   form of a volcano plot ([98]Fig. 2A). The Orthogonal Partial Least
   Squares Discriminant Analysis model score chart effectively
   distinguished between the two sample sets ([99]Fig. 2B). Hierarchical
   cluster analysis revealed significant differences in metabolites
   ([100]Fig. 2C). KEGG pathway enrichment analysis indicated PD-L1
   involvement in metabolism-related pathways, with lipid metabolism
   reprogramming being one of them ([101]Fig. 2D–K).

Fig. 2.

   [102]Fig. 2
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   PD-L1 induces lipid metabolism reprogramming in liver cancer.

   (A) Differential analysis of all detected metabolites (including
   unidentified ones) in positive ion modes based on univariate analysis.
   Volcano graph visualisation of differential metabolites with FC >1.5 or
   FC <0.67 and p <0.05. (B) OPLS-DA score chart in positive ion mode. (C)
   Hierarchical clustering heat map of significant difference metabolites.
   (D) KEGG pathways enrichment. (E–K) Hierarchical clustering heat map
   showing significant differences in glucose metabolites, lipid
   metabolites, nucleotide metabolites, amino acids metabolites, energy
   metabolites, signalling pathways, and neuroactive ligand–receptor
   interaction. Each column represents the mean ± SD of three independent
   experiments. PD-L1 KO denotes PD-L1 knockout cells. ∗Statistically
   significant difference, p <0.05, Student’s t test. FC, fold change;
   KEGG, Kyoto Encyclopedia of Genes and Genomes; KO, knockout; OPLS-DA,
   Orthogonal Partial Least Squares Discriminant Analysis; PD-L1,
   programmed death-ligand 1; WT, wild type.

   Using the shotgun lipidomic analysis of targeted metabolites, we
   extensively identified and quantified the lipid composition of HepG2
   cells. The metabolomics experiments throughout the study identified
   1,019 unique lipid species, including cholesterol ester (chE), coenzyme
   (Co), TG, phosphatidylethanolamine, sphingomyelin, and other sterol
   lipids ([104]Fig. 3A). A volcano plot illustrated changes in the
   expression of different lipid molecules ([105]Fig. 3B), and these lipid
   molecules were mapped and displayed according to their subclasses
   ([106]Fig. 3C–G). PD-L1 KO reduced the number of lipid molecules with
   varying carbon chain lengths and diminished lipid saturation in HepG2
   cells ([107]Fig. 3H–Q). In conclusion, our findings support the
   assertion that PD-L1 contributes to lipid metabolism reprogramming in
   liver cancer.

Fig. 3.

   [108]Fig. 3
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   PD-L1-mediated lipid metabolism reprogramming in liver cancer.

   (A) Lipid subclass content of major lipid species in HepG2 WT and KO
   cell lines analysed by LC-MS/MS. (B) Volcano plot displaying
   differential analysis of all detected lipid molecules in the targeted
   metabolomics of WT vs. PD-L1 KO cells. Rose red dots represent
   significantly different lipids (FC >1.5 or FC <0.67). (C–G) Content of
   major ChE, Co, TG, LPE, and LPS distributions in HepG2 WT and PD-L1 KO
   cells. (H–L) Contents of lipid molecules with different carbon chain
   lengths in each subcategory in HepG2 WT and PD-L1 KO cells. (M–Q)
   Contents of lipid molecules with different numbers of unsaturated bonds
   in each subcategory in HepG2 WT and PD-L1 KO cells. Each column
   represents the mean ± SD of three independent experiments.
   Statistically significant difference, ∗p <0.05, Student’s t test. ChE,
   cholesterol ester; Co, coenzyme; FC, fold change; KO, knockout;
   LC-MS/MS, liquid chromatography with tandem mass spectrometry; LPE,
   lysophosphatidylethanolamine; LPS, lysophosphatidylserine; PD-L1,
   programmed death-ligand 1; TG, triglyceride; WT, wild type.

PD-L1 forms a complex by interacting with EGFR and/or ITGB4

   Next, we endeavoured to elucidate the underlying mechanism by which
   PD-L1 regulates lipid metabolism reprogramming. Given that PD-L1 binds
   to EGFR and influences the sensitivity of gastric cancer cells,[110]^11
   we confirmed the interaction between PD-L1 and EGFR in HepG2 and Huh7
   cells ([111]Fig. S2-1A–F). As cetuximab is a monoclonal antibody that
   binds to EGFR,[112]^23 we assessed the impact of cetuximab on the
   PD-L1–EGFR interaction. Interestingly, cetuximab significantly
   inhibited the interaction between PD-L1 and EGFR in HepG2 and Huh7
   cells ([113]Fig. S2-1A, B, D, and E). The working concentration of
   cetuximab was determined in HepG2 and Huh7 cells ([114]Fig. S2-2A and
   B). However, cetuximab failed to affect EGFR mRNA and protein levels
   ([115]Fig. S2-2C and D). EGFR knockdown or cetuximab treatment
   mitigated the influence of PD-L1 on intracellular TGs and cholesterols,
   whereas EGFR overexpression exhibited the opposite effect
   ([116]Fig. S2-2E–N), suggesting that PD-L1 induces lipid metabolism
   reprogramming by interacting with EGFR in the cells ([117]Fig. S2-2O).

   Given that PD-L1 functions in glucose metabolism reprogramming by
   directly binding to ITGB4,[118]^24 we investigated whether the
   interaction between PD-L1 and ITGB4 contributes to lipid metabolism
   reprogramming. As expected, we confirmed the interaction between PD-L1
   and ITGB4 ([119]Fig. S2-3A–D) and observed that siITGB4 blocked
   PD-L1-mediated intracellular TG and cholesterol accumulation in HepG2
   and Huh7 cells ([120]Fig. S2-3E–J). Conversely, ITGB4 overexpression
   rescued siPD-L1-mediated events ([121]Fig. S2-3K–N). PD-L1 modulates
   lipid metabolism reprogramming by binding to ITGB4 in liver cancer
   cells ([122]Fig. S2-3O).

   Based on positive correlations between PD-L1 and EGFR/ITGB4 in clinical
   samples from 35 HCC donors ([123]Fig. S2-4A), we hypothesised that
   PD-L1, EGFR, and ITGB4 form a complex that modulates the reprogramming
   of lipid metabolism. Immunofluorescence staining demonstrated
   co-localisation of PD-L1/EGFR, PD-L1/ITGB4, EGFR/ITGB4, and
   PD-L1/EGFR/ITGB4 in HepG2 and Huh7 cells ([124]Fig. 4A and
   [125]Fig. S2-4B). Moreover, pull-down assays showed that the purified
   His-tagged PD-L1 extracellular domain protein could be pulled down by
   Fc-EGFR and Fc-ITGB4 extracellular domains (as bait proteins)
   ([126]Fig. 4B). Similar results were observed for the EGFR
   extracellular domain binding to the PD-L1 and ITGB4 extracellular
   domains ([127]Fig. 4C) and for the ITGB4 extracellular domain binding
   to the PD-L1 and EGFR extracellular domains ([128]Fig. 4D). The
   co-localisation of PD-L1, EGFR, and ITGB4 proteins was also validated
   by fluorescence resonance energy transfer (FRET) assays in HepG2 cells
   ([129]Fig. 4E–G). PD-L1 enhanced low-affinity interactions between EGFR
   and ITGB4 ([130]Fig. 4H). Pembrolizumab, a PD-1 antibody, failed to
   bind PD-L1 to EGFR/ITGB4 ([131]Fig. S2-4C and D). We analysed the
   precise interaction sites for the complex through bioinformatics using
   Discovery Studio software. Interestingly, we observed that several
   amino acid residues in the PD-L1 extracellular domain were responsible
   for the interaction between the EGFR and ITGB4 extracellular domains
   (PD-L1, Protein Data Bank ID: 3BIS; EGFR, Protein Data Bank ID: 1IVO;
   ITGB4, predicted) ([132]Fig. S2-5A–F). Accordingly, our results showed
   that the PD-L1 mutants (R125A and D215A) attenuated the binding of EGFR
   to ITGB4 ([133]Fig. S2-5G). Taken together, we concluded that PD-L1
   forms a complex by interacting with EGFR and/or ITGB4 ([134]Fig. 4I).

Fig. 4.

   [135]Fig. 4
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   PD-L1 forms a complex by interacting with EGFR and/or ITGB4.

   (A) Confocal immunofluorescence microscopy in HepG2 cells documented
   the co-localisation of PD-L1, EGFR, and ITGB4 proteins. Scale bar =
   5/2.5 μm. (B) Pull-down assays using Fc antibody in Western blot
   analysis demonstrated PD-L1 binding to purified Fc, Fc-EGFR fusion
   protein, or Fc-ITGB4 fusion protein. (C) Western blot analysis revealed
   EGFR binding to purified Fc, Fc-PD-L1 fusion protein, or Fc-ITGB4
   fusion protein in pull-down assays. (D) Pull-down assays and Western
   blot analysis showed ITGB4 binding to purified Fc, Fc-PD-L1 fusion
   protein, or Fc-EGFR fusion protein. (E–H) FRET assays in HepG2 cells
   validated the co-localisation of PD-L1, EGFR, and ITGB4 proteins with
   indicated treatments. Quantitative FRET measurements (left), and
   corresponding pseudo-colour ED and RC images and their histograms
   (right) are presented. (I) A model depicting the interaction among
   PD-L1, EGFR, and ITGB4 in cancer cells. AA, acceptor-acceptor FRET;
   CFP, cyan fluorescent protein; DA, donor-acceptor FRET; DD, donor-donor
   FRET; E[D], Donor-centric FRET efficiency; EGFR, epidermal growth
   factor receptor; FRET, fluorescence resonance energy transfer; ITGB4,
   integrin β4; PD-L1, programmed death-ligand 1; Rc, acceptor-to-donor
   concentration ratio; YFP, yellow fluorescent protein.

PD-L1 triggers lipid metabolism reprogramming via
EGFR/ITGB4/PI3K/Akt/mTOR/SREBP1c/FASN/ACLY signalling

   PD-L1 promotes cell growth via mTOR signalling in head and neck
   squamous cell carcinomas.[137]^25 The mTOR signalling pathway
   contributes to lipid metabolism by upregulating the expression of
   lipogenic enzymes such as FASN and ACLY.[138]^26^,[139]^27 Therefore,
   we hypothesised that the PD-L1/EGFR/ITGB4 complex promotes the
   reprogramming of lipid metabolism via PI3K/Akt/mTOR/SREBP1c signalling.
   We validated the effects of PD-L1, PD-L1/siEGFR, PD-L1/cetuximab,
   PD-L1/siITGB4, PD-L1/siEGFR/siITGB4, and PD-L1/cetuximab/siITGB4 on
   p-PI3K, p-Akt, and p-mTOR in HepG2 and Huh7 cells ([140]Fig. 5A, B).
   Conversely, treatment with siPD-L1, siPD-L1/EGFR, siPD-L1/ITGB4, and
   siPD-L1/EGFR/ITGB4 showed contrasting results ([141]Fig. 5C and D).
   PD-L1 KO also decreased the levels of p-PI3K, p-Akt, and p-mTOR
   compared with the negative control (NC) group in HepG2 cells
   ([142]Fig. 5E). We used the PI3K inhibitor LY294002, Akt inhibitor
   MK-2206, and mTOR inhibitor rapamycin to validate this hypothesis. As
   expected, PD-L1 overexpression increased FASN and ACLY expression, and
   rapamycin treatment significantly blocked these effects ([143]Fig. 5F).
   Functionally, PD-L1 contributes to lipid metabolism reprogramming by
   activating PI3K/Akt/mTOR/ACLY signalling in liver cancer ([144]Fig. 5G
   and H). We investigated the effects of EGFR and ITGB4 on the
   PI3K/Akt/mTOR pathway ([145]Fig. S3-1A–D). The orthotopic liver mouse
   model showed that treatment with an anti-PD-L1 antibody, but not an
   anti-PD-1 antibody, inhibited tumour growth and reduced the levels of
   tissue TGs and cholesterols, and the Akt inhibitor MK-2206 enhanced
   this effect ([146]Fig. S3-2A–C). PD-L1 knockdown reduced the expression
   of SREBP1c but not PPARs and CCAAT/enhancer-binding protein alpha
   (CEBP-α) in HepG2 cells ([147]Fig. S3-2D–F). Remarkably, PD-L1
   modulated SREBP1c and its downstream lipogenic enzymes FASN and ACLY in
   HepG2 cells via EGFR, ITGB4, and mTOR signalling ([148]Fig. S3-3A).
   siSREBP1c attenuated the PD-L1/EGFR/ITGB4-mediated increase in both TG
   and cholesterol levels ([149]Fig. S3-3B–E). Thus, we conclude that
   PD-L1 triggers lipid metabolism reprogramming by driving
   EGFR/ITGB4/PI3K/Akt/mTOR/SREBP1c/FASN/ACLY signalling in liver cancer.

Fig. 5.

   [150]Fig. 5
   [151]Open in a new tab

   PD-L1 triggers lipid metabolism reprogramming via
   EGFR/ITGB4/PI3K/Akt/mTOR/SREBP1c/FASN/ACLY signalling.

   (A–E) Western blot analysis evaluated the protein levels of total
   PI3K/p-PI3K, total Akt/p-Akt, and total mTOR/p-mTOR in cells with
   indicated treatments. (F) Western blot analysis determined the
   expression levels of FASN and ACLY in HepG2 and Huh7 cells following
   the indicated treatments. (G and H) Tissue total triglyceride assay kit
   or tissue total cholesterol kit measured intracellular triglyceride and
   cholesterol levels in HepG2 and Huh7 cells after treatment.
   Statistically significant differences are indicated: ∗∗p <0.01; ∗∗∗p
   <0.001; ns, no significance; Student's t test. The experiment was
   repeated at least three times. ACLY, ATP citrate lyase; CEBP-α,
   CCAAT/enhancer-binding protein alpha; EGFR, epidermal growth factor
   receptor; FASN, fatty acid synthase; ITGB4, integrin β4; KO, knockout;
   mTOR, mammalian/mechanistic target of rapamycin; PD-L1, programmed
   death-ligand 1; PI3K, phosphoinositide 3-kinase; SREBP1c, sterol
   regulatory element-binding protein-1c.

PD-L1-mediated lipid metabolism reprogramming supports tumour cell
proliferation by EGFR/ITGB4/SREBP1c signalling in vitro

   We investigated the impact of PD-L1-mediated lipid metabolism on cell
   proliferation. PD-L1 overexpression significantly enhanced the
   proliferation of HepG2 and Huh7 cells, as demonstrated by
   3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT)
   assays, EdU incorporation assays, colony formation assays, and flow
   cytometry assays ([152]Fig. 6, [153]Fig. S4-1). The use of siEGFR and
   cetuximab effectively nullified PD-L1-mediated events ([154]Fig. 6A–I).
   The overexpression of EGFR robustly rescued siPD-L1-mediated
   suppression of cell proliferation ([155]Fig. 6B–J). We also assessed
   the role of ITGB4 in PD-L1-mediated lipid metabolism reprogramming for
   modulating cell proliferation in vitro ([156]Fig. S4-1A–J), suggesting
   that PD-L1 promotes cell proliferation via EGFR and/or ITGB4. It has
   been reported that Hippo signalling, regulated by the mevalonate
   pathway, can modulate PD-L1,[157]^28^,[158]^29 and the mevalonate
   pathway is implicated in cholesterol genesis.[159]^30^,[160]^31 Our
   data showed that PD-L1 knockdown reduced the proliferation of HepG2
   cells, and treatment with cholesterol rescued this effect
   ([161]Fig. S4-2A–C). PD-L1 overexpression promoted the proliferation of
   HepG2 cells, as determined by MTT, EdU incorporation, and colony
   formation assays ([162]Fig. S4-3A–C). SREBP1c knockdown effectively
   nullified PD-L1-mediated events ([163]Fig. S4-3A–C). Conversely, PD-L1
   KO decreased the proliferation of HepG2 cells, and SREBP1c
   overexpression rescued PD-L1 KO-mediated events ([164]Fig. S4-4A–C). In
   conclusion, we assert that PD-L1-mediated lipid metabolism
   reprogramming supports cell proliferation via EGFR/ITGB4/SREBP1c
   signalling in vitro.

Fig. 6.

   [165]Fig. 6
   [166]Open in a new tab

   PD-L1-mediated lipid metabolism reprogramming supports tumour cell
   proliferation by EGFR/ITGB4/SREBP1c signalling in vitro.

   (A, B) MTT assays were employed to detect cell proliferation in HepG2
   cells subjected to the indicated treatments. (C–F) Cell proliferation
   was assessed using EdU incorporation assays in HepG2 cells following
   the indicated treatments. Scale bar = 20 μm. (G and H) Colony formation
   assays were conducted to examine HepG2 cell proliferation under the
   indicated treatments. (I and J) Flow cytometry assays were used to
   detect cell proliferation in HepG2 cells with the indicated treatment.
   Statistically significant differences are denoted: ∗p <0.05; ∗∗p <0.01;
   ∗∗∗p <0.001; Student’s t test. The experiment was repeated at least
   three times. EGFR, epidermal growth factor receptor; ITGB4, integrin
   β4; PD-L1, programmed death-ligand 1; MTT,
   3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide; OD,
   optical density; SREBP1c, sterol regulatory element-binding protein-1c.

PD-L1-mediated lipid metabolism reprogramming confers tumour growth via
EGFR/ITGB4/SREBP1c in vivo

   To extend these observations in vivo, we treated male nude mice
   carrying allografts of hepatoma cell lines with subcutaneous
   xenografts. As expected, we found that the overexpression of PD-L1
   remarkably increased the volume and weight of tumours; however,
   siEGFR/siITGB4 and cetuximab treatment blocked PD-L1-enhanced tumour
   growth in mice ([167]Fig. 7A–C and [168]Fig. S5-1A–C). PD-L1 increased
   the expression levels of Ki-67, and both EGFR and ITGB4 were implicated
   in the PD-L1-mediated growth of HCC cells ([169]Fig. 7D and
   [170]Fig. S5-1D). We confirmed that the mRNA expression levels of PD-L1
   increased in the PD-L1 overexpressed group ([171]Fig. 7E and
   [172]Fig. S5-1E). TG and cholesterol levels were markedly reduced in
   tumour tissues from mice treated with PD-L1
   overexpression/cetuximab/siITGB4 ([173]Fig. 7F and G, and
   [174]Fig. S5-1F and G). Interestingly, a similar trend was observed
   with Oil Red O staining ([175]Fig. 7D and [176]Fig. S5-1D).

Fig. 7.

   [177]Fig. 7
   [178]Open in a new tab

   PD-L1-mediated lipid metabolism reprogramming confers tumour growth via
   EGFR/ITGB4/SREBP1c in vivo.

   (A) Photographs depict dissected tumours from nude mice injected with
   HepG2 cells following the indicated treatment (n = 6 mice/group). (B)
   Growth curves illustrate tumour progression in nude mice. (C) Average
   tumour weight is reported for each group. (D) Ki-67 expression in
   tumour tissues from nude mice was assessed by IHC assays. Lipogenesis
   in the tumour tissues from mice transplanted with HepG2 cells was
   determined by Oil Red O staining using frozen sections. (E) PD-L1 mRNA
   expression levels in tumour tissues from mice were determined by
   RT-qPCR analysis. (F and G) Triglyceride or cholesterol levels were
   measured using the tissue triglyceride assay kit or tissue total
   cholesterol kit in the tumour tissues from mice. Statistically
   significant differences are denoted: ∗∗p <0.01; ∗∗∗p <0.001; Student’s
   t test. The experiment was repeated at least three times. EGFR,
   epidermal growth factor receptor; IHC, immunohistochemical; ITGB4,
   integrin β4; OD, optical density; PD-L1, programmed death-ligand 1;
   RT-qPCR, real-time quantitative PCR; SREBP1c, sterol regulatory
   element-binding protein-1c.

   Moreover, we repeated the above experiments in the C57BL/6 orthotopic
   tumour mouse model using Hepa1-6 wild-type (WT) and Hepa1-6 pd-l1 KO
   cells. The obtained results were consistent with the findings in
   immunodeficient mice ([179]Fig. S5-2A–D). PD-L1 overexpression
   increased tumour growth, whereas SREBP1c knockdown attenuated this
   effect in mice ([180]Fig. S5-3A–C). The levels of Ki-67 expression,
   lipid droplets, TGs, and cholesterols were markedly increased in tumour
   tissues from mice overexpressing PD-L1, whereas SREBP1c knockdown
   blocked these effects ([181]Fig. S5-3D–G). However, PD-L1 KO resulted
   in the opposite effect, and SREBP1c overexpression rescued PD-L1
   KO-mediated events ([182]Fig. S5-4). Collectively, we conclude that
   PD-L1-mediated lipid metabolism reprogramming promotes tumour growth
   via EGFR/ITGB4/SREBP1c signalling in vivo.

Discussion

   The expression of PD-L1 by cancer cells serves as a mechanism to evade
   T-cell-mediated immunosurveillance.[183]^32^,[184]^33 In addition,
   cancer cells, characterised by hyperactive growth, use an intricate
   network of lipid metabolic pathways to sustain their burgeoning
   biomass. However, the immune-independent roles of PD-L1, such as
   modulation of lipid metabolism in tumours, are poorly understood. Given
   the limited comprehension of PD-L1, this study aimed to reveal the
   novel roles of PD-L1 in tumorigenesis.

   Analysis of TCGA data revealed that patients with liver cancer and
   displaying elevated PD-L1 levels exhibited diminished survival rates.
   Furthermore, we observed a PD-L1 positivity rate of 80% (64/80) in HCC
   tissues, with significantly heightened PD-L1 mRNA and protein
   expression in liver cancer tissues compared with peritumoral tissues.
   Our investigation also revealed pronounced PD-L1 expression in various
   tumour cell lines. RNA-Seq analysis illustrated that PD-L1 plays a role
   in the biosynthesis of unsaturated FAs, the PPAR signalling pathway,
   and cholesterol metabolism. This finding suggests that PD-L1 actively
   participates in the modulation of lipid metabolic reprogramming in HCC.

   In this study, we illustrated that PD-L1 contributes to the
   accumulation of TGs, cholesterols, and lipid droplets in tumours.
   Lipogenic enzymes critical for tumour growth and alterations, such as
   metabolite abundance and the accumulation of lipid metabolic products,
   play a role in tumour development.[185]^6 Notably, PD-L1 strongly
   modulates the levels of lipid metabolism transcription factors,
   synthases, oxidases, and intracellular lipid metabolism products.
   Comprehensive mass spectrometry-based targeted/untargeted metabolomic
   analysis demonstrated that PD-L1 influences metabolites, including
   lipids, glucose, amino acids, nucleotides, and energy metabolism,
   participating in crucial signalling pathways in HCC cells. This
   observation aligns with the RNA-Seq transcriptome sequence analysis of
   PD-L1 in tumours. Thus, PD-L1 appears to influence lipid metabolism
   reprogramming and modulate multiple metabolic reprogramming processes
   in tumours globally.

   Next, we excavated PD-L1 binding membrane proteins to determine the
   mechanism by which PD-L1 regulates lipid metabolism reprogramming in
   tumours. Interestingly, we found that PD-L1 interacts with EGFR to
   trigger the reprogramming of lipid metabolism in HCC cells. Moreover,
   the EGFR-specific monoclonal antibody cetuximab inhibits lipid
   metabolism reprogramming by blocking the interaction of EGFR with
   PD-L1. Considering that PD-L1 functions in glucose metabolism
   reprogramming by directly binding to ITGB4 in cervical cancer,[186]^24
   we hypothesised that PD-L1 drives lipid metabolism reprogramming by
   interacting with ITGB4 in HCC. These events were observed in these
   cells. We further validated that PD-L1 directly interacts with EGFR and
   ITGB4 to form complexes in cells. This suggests that PD-L1 triggers the
   reprogramming of lipid metabolism in tumours, depending on the complex.
   It is possible that the roles of EGFR and ITGB4 inhibition are
   independent of PD-L1, which is consistent with a previous study that
   EGFR regulates lipid metabolism in liver cancer.[187]^34 It has been
   reported that cytoplasmic PD-L1 plays a critical role in cancer
   development.[188]^35 However, the involvement of cytoplasmic PD-L1 in
   the regulation of lipogenesis in cancer cells remains unclear. Patients
   who respond to the anti-EGFR antibody cetuximab often develop
   resistance to this therapy.[189]^36 Our findings suggest that in the
   event of cetuximab resistance, PD-L1-mediated lipid metabolism
   reprogramming might be involved, providing a new key to understanding
   cetuximab resistance in the clinic. Thus, the anti-PD-L1 antibody might
   release resistance to the anti-EGFR antibody, cetuximab, in cancer
   therapy. Clinically, the application of an anti-PD-L1 antibody might
   inhibit lipid metabolism reprogramming in tumours.

   The mTOR signalling is implicated in the reprogramming of lipid
   metabolism in tumours.[190]^37 We demonstrated that the
   PD-L1/EGFR/ITGB4 complex contributes to lipid metabolism reprogramming
   by activating PI3K/Akt/mTOR/SREBP1c/FASN/ACLY signalling in liver
   cancer. TGs and other fat metabolites influence the activity of PPARs,
   a class of transcription factors that regulate lipid oxidative
   enzymes.[191]^38 Our data suggest that PD-L1-modulated lipid metabolite
   accumulation positively influences lipid metabolism-related enzymes in
   a feedback loop. Given the role of PD-L1 in inhibiting antitumour
   immunity,[192]^39 we hypothesised that PD-L1-mediated lipid metabolism
   reprogramming might be involved in these events. Functionally, we
   validated that PD-L1 promotes the reprogramming of lipid metabolism by
   interacting with EGFR/ITGB4 in vivo. Currently, antibodies targeting
   the PD-L1/PD-1 axis are under evaluation in many clinical trials and
   have been approved for cancer treatment.[193]^39 Our study redefines
   the role of PD-L1 as an immunotherapy target for tumours in the clinic,
   enhancing the understanding of resistance to anti-PD-L1 antibodies in
   cancer therapy. Abnormal lipogenesis is a hallmark of cancer
   cells.[194]^40 In this study, we found that PD-L1-mediated lipid
   metabolism contributes to cancer progression in vitro and in vivo. A
   study in nude mice excluded the major effects of PD-L1 via the immune
   system. Our findings provide new insights into the mechanisms by which
   PD-L1 modulates lipid metabolism reprogramming in tumours.
   Therapeutically, anti-PD-L1 antibodies are available for the treatment
   of lipid metabolism reprogramming in tumours.

Conclusion

   Our findings indicate that tumour cell-expressed PD-L1 initiates the
   reprogramming of lipid metabolism by activating the
   EGFR/ITGB4/PI3K/Akt/mTOR/SREBP1c/FASN/ACLY axis in tumours. In this
   model, PD-L1 induces lipid metabolism reprogramming via the activation
   of EGFR and/or ITGB4, forming a complex with EGFR and ITGB4.
   Subsequently, this complex activation leads to the initiation of
   PI3K/Akt/mTOR/SREBP1c/FASN/ACLY signalling, ultimately resulting in the
   accumulation of lipid metabolites within tumours. PD-L1-mediated lipid
   metabolism reprogramming actively supports tumour progression.

Financial support

   This work was supported by the National Natural Science Foundation of
   China (No. 82372818 to XDZ, No. 82302887 to HFY, No. 82103066 to
   GY, and No. 82303210 to YFW), the China Postdoctoral Science Foundation
   (No. 2022M712389 to HFY, No. 2023M732624 to YFW, and No. 2023M742621 to
   LZ), Tianjin Key Medical Discipline (Specialty) Construction Project
   (TJYXZDXK-009A to WL), and the “14th Five-Year Plan” Tumor Prevention
   and Treatment Research Project of Tianjin Medical University Cancer
   Institute and Hospital (No. YZ-03).

Authors’ contributions

   Designed the experiments: MZ, HFY, XDZ, WL, NNZ. Performed the
   experiments: MZ, HFY, GY. Analysed the data: MZ, HFY, GY. Interpreted
   the results: MZ, HFY, GY. Performed the cell experiments and in vivo
   studies: YFW, YNB, HHZ. Provided the methodology: LNZ, PL, HLY, YG.
   Contributed to the interpretation and conceptual advancement: SW, XDZ,
   WL, NNZ. Wrote the manuscript: MZ, HFY, XDZ. Revised the manuscript:
   GY, XDZ.

Data availability statement

   All data are available upon request from the corresponding author.

Conflicts of interest

   The authors declare no potential Conflicts of interest.

   Please refer to the accompanying ICMJE disclosure forms for further
   details.

Footnotes

   Author names in bold designate shared co-first authorship

   Supplementary data associated with this article can be found, in the
   online version, at [195]https://doi.org/10.1016/j.jhepr.2024.101009.

Contributor Information

   Ningning Zhang, Email: mail4ningning@163.com.

   Wei Lu, Email: mail4luwei@tmu.edu.cn.

   Xiaodong Zhang, Email: zhangxiaodong@tjmuch.com.

Supplementary data

   The following are the supplementary data to this article:
   Multimedia component 1
   [196]mmc1.pdf^ (12.9MB, pdf)
   Multimedia component 2
   [197]mmc2.docx^ (42.1KB, docx)
   Multimedia component 3
   [198]mmc3.pdf^ (2.2MB, pdf)
   Multimedia component 4
   [199]mmc4.pdf^ (19MB, pdf)

References