Programmed Death-ligand 1 Protein Levels Research Articles

Transcription Factor ETV4 Activates AURKA to Promote PD-L1 Expression and Mediate Immune Escape in Lung Adenocarcinoma

Introduction: The occurrence and progression of lung adenocarcinoma (LUAD) impair T-cell immune responses, causing immune escape and subsequently affecting the efficacy of immunotherapy in patients. Aurora kinase A (AURKA) is upregulated in varying cancers, but its role in LUAD immune escape is elusive. This work attempted to explore molecular mechanisms of AURKA regulation in LUAD immune escape. Methods: Through bioinformatics analysis, AURKA level in LUAD was evaluated, and potential upstream transcription factors of AURKA were predicted using hTFtarget. ETS variant transcription factor 4 (ETV4) expression in LUAD was analyzed through The Cancer Genome Atlas. Pearson’s correlation analysis was then utilized to test the correlation between AURKA and ETV4. Interaction and binding between AURKA and ETV4 were validated through dual-luciferase assay and chromatin immunoprecipitation. Quantitative reverse transcription-polymerase chain reaction (qRT-PCR) tested relative mRNA expression of AURKA and ETV4 in LUAD cells, cell counting kit-8 assayed cell viability, and Western blot analysis was conducted to determine the protein level of programmed death-ligand 1 (PD-L1). Coculture of LUAD cells with activated CD8+ T cells was carried out, and an LDH assay was used to assess the cytotoxicity of CD8+ T cells against LUAD cells. Interferon-γ (IFN-γ), interleukin-2 (IL-2), and tumor necrosis factor-α (TNF-α) levels in the coculture system were assessed by enzyme-linked immunosorbent assay (ELISA). Western blot assessed protein levels of JAK2, p-JAK2, STAT3, and p-STAT3. Results: Compared to normal tissues, AURKA and ETV4 were upregulated in tumor tissues, and AURKA presented a negative association with CD8+ T-cell immune infiltration but a positive association with PD-L1. qRT-PCR unveiled significantly upregulated mRNA of AURKA and ETV4 in LUAD cells compared to normal lung epithelial cells. Knockdown of AURKA significantly decreased cell viability and PD-L1 protein level in LUAD cells, enhanced cytotoxicity of CD8+ T cells against LUAD cells and IFN-γ, IL-2, and TNF-α expression, while overexpression of AURKA yielded opposite results. Furthermore, the knockdown of ETV4 could reverse the oncogenic characteristics of cells caused by AURKA overexpression. Conclusion: Our study illustrated that ETV4/AURKA axis promoted PD-L1 expression, suppressed CD8+ T-cell activity, and mediated immune escape in LUAD by regulating the JAK2/STAT3 signaling pathway.

Acesulfame potassium upregulates PD-L1 in HCC cells by attenuating autophagic degradation

Artificial sweeteners, which contain no or few calories, have been widely used in various foods and beverages, and are regarded as safe alternatives to sugar by the Food and Drug Administration. While several studies suggest that artificial sweeteners are not related to cancer development, some research has reported their potential association with the risk of cancers, including hepatocellular carcinoma (HCC). Here, we investigated whether acesulfame potassium (Ace K), a commonly used artificial sweetener, induces immune evasion of HCC cells by upregulating programmed death ligand-1 (PD-L1). Ace K elevated the protein levels of PD-L1 in HCC cells without increasing its mRNA levels. The upregulation of PD-L1 protein levels in HCC cells by Ace K was induced by attenuated autophagic degradation of PD-L1, which was mediated by the Ace K-stimulated ERK1/2-mTORC1 signaling pathway. Ace K-induced upregulation of PD-L1 attenuated T cell-mediated death of HCC cells, thereby promoting immune evasion of HCC cells. In summary, the present study suggests that Ace K promotes HCC progression by upregulating the PD-L1 protein level.

Chromosome 9p Duplication Promotes T-Cell Exhaustion and Enhances Stem Cell Clonogenic Potential in JAK2-Mutant Myeloproliferative Neoplasms

Myeloproliferative Neoplasms (MPNs) are a diverse group of clonal hematopoietic disorders originating from a single hematopoietic stem cell, causing an excessive production of mature blood cells. Classic forms of MPNs include polycythemia vera, essential thrombocythemia, and myelofibrosis. The most common mutation is a gain-of-function point mutation in the JAK2 gene, known as JAK2V617F. This mutation leads to constant activation of the JAK-STAT signaling pathway, resulting in the overgrowth of MPN cells. Furthermore, the variant allele frequency (VAF) of JAK2V617F has a significant impact on the severity and phenotype of the disease. Alongside many described genetic mutations, cytogenetic abnormalities are commonly observed in MPNs, particularly involving chromosome 9. As the JAK2 gene is located on the short arm of this chromosome, we hypothesized that chromosome 9 copy number abnormalities might be a disease modifier in JAK2V617F-mutant MPN patients. To characterize the biological effects of chromosome 9 copy number abnormalities on JAK2-mutated MPN cells, we analyzed circulating CD34+ hematopoietic stem and progenitor cells (HSPCs) as well as monocytes and granulocytes from 32 MPN patients. Through Next Generation Sequencing, we categorized the patients into three main groups based on JAK2 mutation and copy number status: patients with two copies of the JAK2 gene with either heterozygous (VAF between 20% and 60%; n=10) or homozygous (VAF > 60% n=10) JAK2V617F mutation, or patients carrying mutant JAK2 and gene amplification (n=12). In-depth analysis of JAK2-amplified patients revealed that the amplification involved the entire chromosome 9p, thus including other gene loci like CD274, which encodes programmed death-ligand 1 (PD-L1). Further investigation of the order-of-events and clonal hierarchies through droplet digital PCR on CD34+ cell-derived colonies showed that most colonies of 9p-duplicated patients had three copies of JAK2, with 2 out of 3 alleles harboring the JAK2 mutation and that point mutations are frequently the initial pathogenic event in clonal evolution, followed by amplification of the JAK2-mutated allele. Functionally, CD34+ cells from +9p patients displayed high clonogenicity and gave rise to a greater number of primitive colonies (Colony Forming Unit-Granulocyte, Erythrocyte, Monocyte, Megakaryocyte, CFU-GEMM). As JAK2 hyperactivation had been previously reported to lead to increased PD-L1 expression, we further explored the functional significance of CD274 amplification in +9p patients. Our analysis showed increased PD-L1 messenger RNA and protein levels in +9p patient CD14+ monocytes compared to those from patients carrying only two copies of chromosome 9. Moreover, immunofluorescence analysis demonstrated significant re-localization of PD-L1 to the cytoplasmic membrane in monocytes from +9p patients, but not in JAK2V617F-homozygous patients (panel A). Increased levels of PD-L1, an immune checkpoint known to curb T cell activation, led us to analyze the T cell compartment, which resulted enriched in CD3+/CD8+/CD57-/PD-1+ exhausted T-cells in +9p patients compared to other MPN patients and healthy donors (panel B). In conclusion, our comprehensive characterization of the molecular interplay between JAK2V617F and chromosome 9 alterations, along with their immunological implications due to PD-L1 hyperactivation, fills a critical knowledge gap and provides valuable insights into the disease progression of 9p-MPNs. Further analysis is ongoing to explore the associations between 9p duplication and hematological parameters in 9p-MPN patients for a better understanding of the clinical implications of this genetic abnormality in MPNs.

Astragaloside IV Attenuates Programmed Death-Ligand 1-Mediated Immunosuppression during Liver Cancer Development via the miR-135b-5p/CNDP1 Axis.

Astragaloside IV (AS-IV) is a pivotal contributor to anti-tumour effects and has garnered extensive attention in research. Tumour cell immune suppression is closely related to the increase in Programmed Death-Ligand 1 (PD-L1). Hepatocellular carcinoma (HCC) is a malignant tumour originating from hepatic epithelial tissue, and the role of AS-IV in regulating PD-L1 in anti-HCC activity remains unclear. Various concentrations of AS-IV were administered to both human liver immortalised cells (THEL2) and HCC (Huh-7 and SMMC-7721), and cell growth was assessed using the CCK-8 assay. HCC levels and cell apoptosis were examined using flow cytometry. Mice were orally administered AS-IV at different concentrations to study its effects on HCC in vivo. Immunohistochemistry was employed to evaluate PD-L1 levels. Western blotting was employed to determine PD-L1 and CNDP1 protein levels. We carried out a qRT-PCR to quantify the levels of miR-135b-3p and CNDP1. Finally, a dual-luciferase reporter assay was employed to validate the direct interaction between miR-135b-3p and the 3'UTR of CNDP1. AS-IV exhibited a dose-dependent inhibition of proliferation in Huh-7 and SMMC-7721 while inhibiting PD-L1 expression induced by interferon-γ (IFN-γ), thus attenuating PD-L1-mediated immune suppression. MiR-135b-5p showed significant amplification in HCC tissues and cells. AS-IV mitigated PD-L1-mediated immune suppression through miR-135b-5p. MiR-135b-5p targeted CNDP1, and AS-IV mitigated PD-L1-induced immunosuppression by modulating the miR-135b-5p/CNDP1 pathway. AS-IV decreases cell surface PD-L1 levels and alleviates PD-L1-associated immune suppression via the miR-135b-5p/CNDP1 pathway. AS-IV may be a novel component for treating HCC.

PPARγ inhibited tumor immune escape by inducing PD-L1 autophagic degradation.

Blockade of the programmed death 1 (PD-1)/ programmed death ligand 1 (PD-L1) immune checkpoint could increase antitumor immunotherapy for multiple types of cancer, but the response rate of patients is about 10%-40%. Peroxisome proliferator activated receptor γ (PPARγ) plays an important role in regulating cell metabolism, inflammation, immunity, and cancer progression, while the mechanism of PPARγ on cancer cell immune escape is still unclear. Here we found that PPARγ expression exhibits a positive correlation with activation of T cells in non-small-cell lung cancer (NSCLC) by clinical analysis. Deficiency of PPARγ promoted immune escape of NSCLC by inhibiting T-cell activity, which was associated with increased PD-L1 protein level. Further analysis showed that PPARγ reduced PD-L1 expression independent of its transcriptional activity. PPARγ contains the microtubule-associated protein 1A/1B-light chain 3 (LC3) interacting region motif, which acts as an autophagy receptor for PPARγ binding to LC3, leading to degradation of PD-L1 in lysosomes, which in turn suppresses NSCLC tumor growth by increasing T-cell activity. These findings suggest that PPARγ inhibits the tumor immune escape of NSCLC by inducing PD-L1 autophagic degradation.

AXL/PLCγ1/PKCα as a Central Axis for PD‐L1 Regulation in Triple‐Negative Breast Cancer

Breast cancer is the most prevalent malignancy in women globally, accounting for 12% of all new annual cancer cases worldwide. Triple-negative breast cancer (TNBC), which is ER, PR and HER2 negative, represents 10-15% of breast cancers and displays an aggressive metastatic phenotype, with limited treatment options and poor patient prognosis. TNBC is associated with high expression levels of immune checkpoint programmed-death ligand 1 (PD-L1) compared with other breast cancer subtypes, and it is therefore amenable to immune checkpoint therapy. In this study, we analyzed the role of diacylglycerol (DAG)-regulated protein kinase C alpha (PKCα) in the control of PD-L1 expression in TNBC. Dataset analysis of 50 breast cancer cell lines revealed a prominent up-regulation of PKCα in TNBC, particularly in TNBC-B subtype, with a concomitant down-regulation of protein kinase C delta (PKCδ). This finding was authenticated in the TCGA-BRCA database. A significant correlation was found between PKCα expression and EMT markers, i.e., up-regulation of vimentin, Zeb1, TWIST1, SNAI2 and AXL, and E-cadherin down-regulation. High PKCα expression was also observed in TNBC patient-derived xenografts (PDXs). Immunofluorescence analysis revealed significant PKCα endogenous levels in the TNBC B cell line BT549, with strong "peripheral staining", a hallmark of its activated state. Conversely, a tenuous PKCα staining was detected in luminal breast cancer cells. Notably, TNBC-B cell lines express high levels of phosphorylated (active)-phospholipase C gamma 1 (PLCγ1), an enzyme responsible for DAG generation and PKCα activation. We also found a significant positive correlation between PKCα and PD-L1 expression in breast cancer cell lines. Notably, both RNAi silencing and pharmacological inhibition of PKCα or PLCγ1 reduced PD-L1 mRNA and protein levels in TNBC cells. We also show that AXL and PKCα mutually and positively control their expression in addition to up-regulating PD-L1, and they share a common gene expression signature that is highly enriched in cytokine, extracellular matrix, and motility pathways. Mechanistic analysis showed that inhibition of AXL, PLCγ1 or PKCα significantly reduced PD-L1 promoter activity in TNBC cells, suggesting a transcriptional control of the PDL1 (CD274) gene by this pathway. In summary, our results indicate that aberrant overactivation of AXL/PLCγ1/PKCα represents a cancer cell intrinsic mechanism for PD-L1 up-regulation in TNBC, suggesting a potential role for this axis in the control of tumor immunosurveillance. PKCα and AXL may be promising candidates for integrated targeted therapy in TNBC treatment.

Interferon‐γ Induced PD‐L1 Expression in Ovarian Cancer Cells is Regulated by IRF1 Signaling

The immune checkpoint programmed death ligand‐1 (PD‐L1) is highly expressed in cancer cells, including ovarian cancer (OC) cells. High PD‐L1 expression in ovarian cancer correlates with poor prognosis, but the mechanisms that regulate the PD‐L1 expression in OC remain poorly understood. We have recently shown that IFNγ induces the PD‐L1 expression in OC cells, resulting in their increased proliferation and invasion. Here, we show that the IFNγ‐induced PD‐L1 expression in OC cells is associated with increased levels of STAT1, Tyr‐701 pSTAT1 and Ser‐727 pSTAT1 in OC cells. JAK1 and STAT1 suppression significantly decreases the IFNγ‐induced PD‐L1 mRNA and protein levels in OC cells. In addition, IRF1 suppression significantly decreases the IFNγ‐induced gene and protein levels of PD‐L1, indicating that the IFNγ‐induced PD‐L1 expression in OC cells is dependent on IRF1 signaling. Chromatin immunoprecipitation data demonstrate that IFNγ induces occupancy of STAT1, Ser‐727 pSTAT1 and IRF1 at STAT1 and IRF1 binding sites in human PD‐L1 promoter. Together, these results show that the IFNγ‐induced PD‐L1 expression in OC cells is regulated by IRF1/JAK1/STAT1 signaling, and suggest that targeting the IRF1/JAK1/STAT1 pathway may provide leverage to regulate the PD‐L1 levels in ovarian cancer.

Dual-sgRNA CRISPR/Cas9 knockout of PD-L1 in human U87 glioblastoma tumor cells inhibits proliferation, invasion, and tumor-associated macrophage polarization

Programmed death ligand 1 (PD-L1) plays a key role in glioblastoma multiforme (GBM) immunosuppression, vitality, proliferation, and migration, and is therefore a promising target for treating GBM. CRISPR/Cas9-mediated genomic editing can delete both cell surface and intracellular PD-L1. This systemic deliverable genomic PD-L1 deletion system can be used as an effective anti-GBM therapy by inhibiting tumor growth and migration, and overcoming immunosuppression. To target PD-L1 for CRISPR/Cas9 gene editing, we first identified two single guide RNA (sgRNA) sequences located on PD-L1 exon 3. The first sgRNA recognizes the forward strand of human PD-L1 near the beginning of exon 3 that allows editing by Cas9 at approximately base pair 82 (g82). The second sgRNA recognizes the forward strand of exon 3 that directs cutting at base pair 165 (g165). A homology-directed repair template (HDR) combined with the dual-sgRNAs was used to improve PD-L1 knockout specificity and efficiency. sgRNAs g82 and g165 were cloned into the multiplex CRISPR/Cas9 assembly system and co-transfected with the HDR template in human U87 GBM cells (g82/165 + HDR). T7E1 analysis suggests that the dual-sgRNA CRISPR/Cas9 strategy with a repair template was capable of editing the genomic level of PD-L1. This was further confirmed by examining PD-L1 protein levels by western blot and immunofluorescence assays. Western blot analysis showed that the dual-sgRNAs with the repair template caused a 64% reduction of PD-L1 protein levels in U87 cells, while immunostaining showed a significant reduction of intracellular PD-L1. PD-L1 deletion inhibited proliferation, growth, invasion and migration of U87 cells, indicating intracellular PD-L1 is necessary for tumor progression. Importantly, U87 cells treated with g82/165 + HDR polarized tumor-associated macrophages (TAM) toward an M1 phenotype, as indicated by an increase in TNF-α and a decrease in IL-4 secretions. This was further confirmed with flow cytometry that showed an increase in the M1 markers Ly6C + and CD80 +, and a decrease in the M2 marker CD206 + both in vitro and in vivo. Utilizing dual-sgRNAs and an HDR template with the CRISPR/Cas9 gene-editing system is a promising avenue for the treatment of GBM.

PD-L1 promotes myofibroblastic activation of hepatic stellate cells by distinct mechanisms selective for TGF-β receptor I versus II.

SUMMARYIntrahepatic cholangiocarcinoma (ICC) contains abundant myofibroblasts derived from hepatic stellate cells (HSCs) through an activation process mediated by TGF-β. To determine the role of programmed death-ligand 1 (PD-L1) in myofibroblastic activation of HSCs, we disrupted PD-L1 of HSCs by shRNA or anti-PD-L1 antibody. We find that PD-L1, produced by HSCs, is required for HSC activation by stabilizing TGF-β receptors I (TβRI) and II (TβRII). While the extracellular domain of PD-L1 (amino acids 19–238) targets TβRII protein to the plasma membrane and protects it from lysosomal degradation, a C-terminal 260-RLRKGR-265 motif on PD-L1 protects TβRI mRNA from degradation by the RNA exosome complex. PD-L1 is required for HSC expression of tumor-promoting factors, and targeting HSC PD-L1 by shRNA or Cre/loxP recombination suppresses HSC activation and ICC growth in mice. Thus, myofibroblast PD-L1 can modulate the tumor microenvironment and tumor growth by a mechanism independent of immune suppression.

Sensitizing tumors to anti-PD-1 therapy by promoting NK and CD8+ T cells via pharmacological activation of FOXO3

BackgroundStimulating antitumor immunity by blocking programmed death-1 (PD-1) or its ligand (programmed death-ligand 1 (PD-L1) is a promising antitumor therapy. However, numerous patients respond poorly to PD-1/PD-L1 blockade. Unresponsiveness to...

Anti-Tumor Effects of Carrimycin and Monomeric Isovalerylspiramycin I on Hepatocellular Carcinoma in Vitro and in Vivo.

Hepatocellular carcinoma results in a high risk of second primary malignancies and has prominent morbidity and mortality. There is a lack of effective treatment and prognosis is poor. Therefore, effective drugs need to be discovered. Carrimycin is a 16-member macrolide antibiotic with anticancer activity, and monomeric isovalerylspiramycin I is a main component. The aim of this study was to determine the anti-tumor effects of carrimycin and monomeric isovalerylspiramycin I on hepatocellular carcinoma through in vivo and in vitro experiments. In vitro, changes in cellular proliferation, migration, invasion, and apoptosis were analyzed by MTT, colony formation, EdU labeling, wound-healing, matrigel transwell invasion, and flow cytometric assays using SK-Hep1, Hep3B, SNU-354, SNU-387 hepatocellular carcinoma cell lines. Western blotting and RT-PCR were used to detect the effects of carrimycin and monomeric isovalerylspiramycin I on the expression levels of vascular endothelial growth factor (VEGF) and programmed death ligand 1 (PD-L1). Nude mice were subcutaneously transplanted with SK-Hep1 cells or C57BL/6J mice were orthotopically transplanted with hepatocarcinoma H22 cells. Tumor volume, pathological changes in tumor tissues, and the concentration of VEGF in mouse serum were measured after treatments. Carrimycin and monomeric isovalerylspiramycin I dose-dependently inhibited hepatocellular carcinoma cell viability, colony formation, and DNA replication. These agents markedly suppressed migration and invasion and promoted apoptosis of the cell lines. Western blotting and RT-PCR demonstrated that carrimycin and monomeric isovalerylspiramycin I reduced VEGF and PD-L1 protein and mRNA levels in a dose-dependent manner. In vivo studies further confirmed that carrimycin and monomeric isovalerylspiramycin I could significantly inhibit tumor growth, tumor histopathological alterations, and the concentration of VEGF in both mouse tumor models. These results show that carrimycin and monomeric isovalerylspiramycin I promoted apoptosis and inhibited proliferation, migration, and invasion of hepatocellular carcinoma cells. Therefore, our discovery suggests anti-tumor capacity for carrimycin and monomeric isovalerylspiramycin I and provides data on potential new drugs for inhibiting hepatocellular carcinoma.

The effect of hypoxia on PD-L1 expression in bladder cancer

IntroductionRecent data has demonstrated that hypoxia drives an immunosuppressive tumour microenvironment (TME) via various mechanisms including hypoxia inducible factor (HIF)-dependent upregulation of programmed death ligand 1 (PD-L1). Both hypoxia and an immunosuppressive TME are targetable independent negative prognostic factors for bladder cancer. Therefore we sought to investigate whether hypoxia is associated with upregulation of PD-L1 in the disease.Materials and methodsThree human muscle-invasive bladder cancer cell lines (T24, J82, UMUC3) were cultured in normoxia (20% oxygen) or hypoxia (1 and 0.1% oxygen) for 24 h. Differences in PD-L1 expression were measured using Western blotting, quantitative polymerase chain reaction (qPCR) and flow cytometry (≥3 independent experiments). Statistical tests performed were unpaired t tests and ANOVA. For in silico work an hypoxia signature was used to apply hypoxia scores to muscle-invasive bladder cancers from a clinical trial (BCON; n = 142) and TCGA (n = 404). Analyses were carried out using R and RStudio and statistical tests performed were linear models and one-way ANOVA.ResultsWhen T24 cells were seeded at < 70% confluence, there was decreased PD-L1 protein (p = 0.009) and mRNA (p < 0.001) expression after culture in 0.1% oxygen. PD-L1 protein expression decreased in both 0.1% oxygen and 1% oxygen in a panel of muscle-invasive bladder cancer cells: T24 (p = 0.009 and 0.001), J82 (p = 0.008 and 0.013) and UMUC3 (p = 0.003 and 0.289). Increasing seeding density decreased PD-L1 protein (p < 0.001) and mRNA (p = 0.001) expression in T24 cells grown in both 20 and 1% oxygen. Only when cells were 100% confluent, were PD-L1 protein and mRNA levels higher in 1% versus 20% oxygen (p = 0.056 and p = 0.037). In silico analyses showed a positive correlation between hypoxia signature scores and PD-L1 expression in both BCON (p = 0.003) and TCGA (p < 0.001) cohorts, and between hypoxia and IFNγ signature scores (p < 0.001 for both).ConclusionTumour hypoxia correlates with increased PD-L1 expression in patient derived bladder cancer tumours. In vitro PD-L1 expression was affected by cell density and decreased PD-L1 expression was observed after culture in hypoxia in muscle-invasive bladder cancer cell lines. As cell density has such an important effect on PD-L1 expression, it should be considered when investigating PD-L1 expression in vitro.

Sorting nexin 6 interacts with Cullin3 and regulates programmed death ligand 1 expression.

Programmed death ligand 1 (PD-L1) is critical for the ability of cancer cells to evade attacks by the host immune system. However, the molecular mechanisms controlling PD-L1 expression have not been fully understood. Here, we demonstrate that sorting nexin 6 (SNX6) is a novel regulator of PD-L1 expression. Knockdown of SNX6 in cancer cells significantly decreases PD-L1 protein levels. In contrast, loss of SNX6 does not reduce PD-L1 mRNA levels. Instead, SNX6 interacts with Cullin3, an E3 ubiquitin ligase responsible for PD-L1 ubiquitination and subsequent degradation. By binding with Cullin3, SNX6 decreases the interaction between the adaptor protein speckle-type POZ protein and Cullin3, which in turn downregulates Cullin3-mediated PD-L1 ubiquitination. This research reveals a novel molecular nexus in modulating PD-L1.

Inhibition of GFAT1 in lung cancer cells destabilizes PD-L1 protein.

Immunotherapy using checkpoint blockers (antibodies) has been a major advance in recent years in the management of various types of solid cancers including lung cancer. One target of checkpoint blockers is programmed death ligand 1 (PD-L1) expressed by cancer cells, which engages programmed death 1 on T cells and Natural Killer (NK) cells resulting in suppression of their activation and cancer-killing function, respectively. Apart from antibodies, other clinically relevant agents that can inhibit PD-L1 are limited. PD-L1 protein stability depends on its glycosylation. Here we show that l-glutamine:d-fructose-6-phosphate amidotransferase 1 (GFAT1), a rate-limiting enzyme of the hexosamine biosynthesis pathway, which produces uridine diphosphate-N-acetyl-β-glucosamine, a precursor for glycosylation, is required for the stability of PD-L1 protein. Inhibition of GFAT1 activity markedly reduced interferon gamma (IFNγ)-induced PD-L1 levels in various lung cancer cell lines. GFAT1 inhibition suppressed glycosylation of PD-L1 and accelerated its proteasomal degradation. Importantly, inhibition of GFAT1 in IFNγ-treated cancer cells enhanced the activation of T cells and the cancer-killing activity of NK cells. These findings support using GFAT1 inhibitors to manipulate PD-L1 protein level that could augment the efficacy of immunotherapy for lung cancer.

LPS stimulates gingival fibroblasts to express PD-L1 via the p38 pathway under periodontal inflammatory conditions

ObjectiveThe overall aim of this research was to investigate the differences in the expression of programmed death ligand 1 (PD-L1) in human gingival fibroblasts (HGFs) between a periodontal healthy group and a periodontal inflammatory group. and explore the possible mechanism involved. MethodsDifferences in PD-L1 mRNA and protein expression in HGFs from a periodontal healthy group and a periodontal inflammatory group were examined by qPCR and western blotting, respectively, and were further tested after lipopolysaccharide (LPS) stimulation in both groups. The effects of a p38 pathway inhibitor on the changes in p38 phosphorylation levels and PD-L1 expression after LPS stimulation were investigated in both groups. ResultsPD-L1 mRNA and protein levels in HGFs in the periodontal inflammatory group were significantly higher than those in the periodontal healthy group (p < 0.05). After 10 μg/mL LPS stimulation, PD-L1 mRNA levels in HGFs from both groups increased significantly (p < 0.05), peaking at 4 h, and the peak was significantly higher in the periodontal inflammatory group than in the periodontal healthy group (p < 0.05). However, PD-L1 protein expression was upregulated only in the inflammatory group (p < 0.05). Inhibition of the p38 pathway in HGFs decreased p38 phosphorylation in both groups (p < 0.05) but this treatment reversed the LPS-induced increase in PD-L1 mRNA and protein levels only in the inflammatory group (p < 0.05). ConclusionIn the periodontal inflammatory state, the expression of PD-L1 in HGFs is more easily activated, and may be influenced by the p38 pathway.

Deubiquitinating enzyme OTUB1 promotes cancer cell immunosuppression via preventing ER-associated degradation of immune checkpoint protein PD-L1.

Upregulation of programmed death ligand 1 (PD-L1) helps tumor cells escape from immune surveillance, and therapeutic antibodies targeting PD-1/PD-L1 have shown better patient outcomes only in several types of malignancies. Recent studies suggest that the clinical efficacy of anti-PD-1/PD-L1 treatments is associated with PD-L1 levels; however, the underlying mechanism of high PD-L1 protein levels in cancers is not well defined. Here, we report that the deubiquitinase OTUB1 positively regulates PD-L1 stability and mediates cancer immune responses through the PD-1/PD-L1 axis. Mechanistically, we demonstrate that OTUB1 interacts with and removes K48-linked ubiquitin chains from the PD-L1 intracellular domain in a manner dependent on its deubiquitinase activity to hinder the degradation of PD-L1 through the ERAD pathway. Functionally, depletion of OTUB1 markedly decreases PD-L1 abundance, reduces PD-1 protein binding to the tumor cell surface, and causes increased tumor cell sensitivity to human peripheral blood mononuclear cells (PBMCs)-mediated cytotoxicity. Meanwhile, OTUB1 ablation-induced PD-L1 destabilization facilitates more CD8+ T cells infiltration and increases the level of IFN-γ in serum to enhance antitumor immunity in mice, and the tumor growth suppression by OTUB1 silencing could be reversed by PD-L1 overexpression. Furthermore, we observe a significant correlation between PD-L1 abundance and OTUB1 expression in human breast carcinoma. Our study reveals OTUB1 as a deubiquitinating enzyme that influences cancer immunosuppression via regulation of PD-L1 stability and may be a potential therapeutic target for cancer immunotherapy.

The Progress of Immunotherapy in Refractory Pituitary Adenomas and Pituitary Carcinomas

Most pituitary adenomas (PAs) are considered benign tumors, but approximately 0.2% can present metastasis and are classified as pituitary carcinomas (PCs). Refractory PAs lie between benign adenomas and true malignant PC and are defined as aggressive-invasive PAs characterized by a high Ki-67 index, rapid growth, frequent recurrence, and resistance to conventional treatments, including temozolomide. It is notoriously difficult to manage refractory PAs and PC because of the limited therapeutic options. As a promising therapeutic approach, cancer immunotherapy has been experimentally used for the treatment of many tumors, including pituitary tumors. The purpose of this review is to report the progress of immunotherapy in pituitary tumors, including refractory PAs and PCs. The tumor immune microenvironment has been recognized as a key contributor to tumorigenesis, progression, and prognosis. One study indicated that the number of CD68+ macrophages was positively correlated with tumor size and Knosp classification grade for tumor invasiveness. The infiltration of CD4+ and CD8+ T cells was relatively scant in these adenomas, but pituitary growth hormone (GH) adenomas exhibited significantly more CD4+ and CD8+ T cells than non-GH adenomas. These results suggest an association of CD68+ macrophage infiltration with an increase in pituitary tumor size and invasiveness. Another study suggested that a lower number of CD8+ lymphocytes is associated with cavernous sinus invasion and resistance to treatment with first-generation somatostatin analogs in acromegaly patients, highlighting a potential role of the tumor immune microenvironment in determining the prognosis of somatotroph pituitary tumors. Preclinical studies have indicated that widely varying degrees of programmed death-ligand 1 (PD-L1) expression and tumor-infiltrating lymphocytes (TILs) are found among different subtypes. Functional PAs and aggressive PAs express significantly higher levels of PD-L1 and TILs than other subtypes, indicating that PD-1 blockade might be a promising alternative therapy for patients with aggressive PAs. PD-L1 transcript and protein levels were found to be significantly increased in functioning (GH and prolactin-expressing) pituitary tumors compared to nonfunctioning (null cell and silent gonadotroph) adenomas. Moreover, primary pituitary tumors harbored higher levels of PD-L1 mRNA than recurrent tumors. These findings suggest the possibility of considering checkpoint blockade immunotherapy for functioning pituitary tumors refractory to conventional management. Animal models of Cushing’s disease also demonstrated PD-L1 and TIL expression in cultured tumors and murine models, as well as the effectiveness of checkpoint blockade therapy in reducing the tumor mass, decreasing hormone secretion, and increasing the survival rate. Clinical studies show that immunotherapy may be an effective treatment in patients with pituitary tumors. One corticotroph carcinoma patient showed a significant reduction in hormone levels and shrinkage of the tumor size of primary and metastatic lesions immediately after investigational treatment with ipilimumab and nivolumab. However, another patient with corticotroph adenoma progressed rapidly after four cycles of anti-PD-1 (pembrolizumab) treatment. To date, there are two registered clinical trials of immunotherapy for pituitary tumors. One of them is the phase II clinical trial of nivolumab combined with ipilimumab for patients with aggressive pituitary tumors (NCT04042753). The other one is also a phase II clinical trial of the combination of nivolumab and ipilimumab for rare tumors, including pituitary tumors (NCT02834013). Both clinical trials are in the stage of recruiting patients and have not been completed. In summary, the results from preclinical research and clinical studies indicated that immunotherapy might be a promising alternative therapy for PCs and refractory PAs resistant to conventional treatments. The combination of immunotherapy and radiotherapy or temozolomide may have synergistic effects compared to a single treatment. More preclinical and clinical studies are needed to further indicate the exact efficacy of immunotherapy in pituitary tumors.

Abstract 6011: Mesenchymal stem cells promote PD-L1 expression in lung cancer cells

Abstract Background Effects of mesenchymal stem cells (MSCs) on tumors have been extensively studied in recent years, but results still remain controversial. In this study we investigated the roles of MSCs on the expression of programmed death-ligand 1 (PD-L1) in lung cancer cells. Since overexpression of PD-L1 has been found to attenuate body's immune system against tumor cells, PD-1/PD-L1 blockages have become the most effective immunotherapy to the late stage non-small cell lung cancer (NSCLC). Therefore, understanding the effects of MSCs on PD-L1 expression is critical in managing immunotherapy for NSCLC patients. Method PD-L1 expressions were tested in two NSCLC cell lines, A549 and H1299 using qRT-PCR and Western Blot to qualitatively measure mRNA and protein levels, respectively. MSCs were isolated from umbilical cord blood and co-cultured with A549 and H1299 cells in complete medium using Transwell filters for 24-h, 48-h and 72-h. Then both A549 and H1299 cells were collected and PD-L1 expressions were tested. MSC-Conditioned Medium (MSC-CM) was generated by culturing 80% confluence of MSCs with serum-free medium. After 48 h, the medium was collected as MSC-CM. Both A549 and H1299 were then cultured in MSC-CM for 24-h, 48-h and 72-h, and PD-L1 expressions were measured from these two cell lines separately. Result Both MSCs and MSC-CM significantly increased the PD-L1 mRNA levels in the two NSCLC cells, while the MSC-CM had stronger stimulation to PD-L1 expressions (P &amp;lt; 0.0001) comparing to MSCs treatment (P&amp;lt;0.01). A549 cells reached the most significant effect at 48-h, while H1299 cells attended the same effect at 24-h treatment. The similar results were observed with PD-L1 protein levels. Both cell lines showed increased PD-L1 protein expressions after treating with MSCs and MSC-CM, but the augmented scales were smaller than the mRNA levels. The PD-L1 protein expressions in A549 cells increased significantly (p &amp;lt; 0.05) at 48-h when treating with MSCs, while increased much higher when treating with MSC-CM (P &amp;lt; 0.01). H1299 cells also showed the similar increase in PD-L1 protein expressions after 24-h treatment. Conclusion Both MSCs and MSC-CM significantly increase PD-L1 expressions in lung cancer cells, which ultimately attenuate the immune system against tumor cells. Citation Format: Hui Zhao, Hui Yang, Jinqiu Sun, Guoqiang Liang, Qianqian Zhao, Xiufang Zhang, Shengxian Liang, Rui Guo, Li Zhong. Mesenchymal stem cells promote PD-L1 expression in lung cancer cells [abstract]. In: Proceedings of the Annual Meeting of the American Association for Cancer Research 2020; 2020 Apr 27-28 and Jun 22-24. Philadelphia (PA): AACR; Cancer Res 2020;80(16 Suppl):Abstract nr 6011.

Pentamethylquercetin Inhibits Hepatocellular Carcinoma Progression and Adipocytes-induced PD-L1 Expression via IFN-γ Signaling.

Obesity is a significant risk factor for the development of types of cancer. Programmed death 1 and its ligand programmed death-ligand 1 (PD-L1) play a crucial role in tumor immune escape. Although, the role of PD-L1 in obesity-associated hepatocellular carcinoma (HCC) remains unknown. We previously showed that the natural flavonoid pentamethylquercetin (PMQ) possesses anti-obesity properties. This study was designed to investigate the effects of PMQ on the development of HCC in obese mice and whether PMQ regulates PD-L1 and expression in HCC. Monosodium glutamate-induced obese mice were inoculated with H22 tumor cells. Tumor volumes and weights were measured. In vitro, 3T3-L1 preadipocytes were differentiated and lipid accumulation was measured by oil-red staining, and IFN-γ level was detected by Elisa. Hepatoma HepG2 cells were treated with conditional media from 3T3-L1 adipocytes (adi-CM). Western blotting was applied to detect PD-L1 protein levels in tumor tissue and HepG2 cells. Compared with control mice, H22 tumors grew faster and exhibited higher PD-L1 protein levels in obese mice. PMQ inhibited H22 tumor growth and reduced PD-L1 expression in tumor tissues. PD-L1 protein level was elevated in adi-CM-treated HepG2 cells. IFN-γ was detectable in adi-CM and exogenous IFN-γ induced PD-L1 expression in HepG2 cells. PMQ affected the differentiation of 3T3-L1 preadipocytes, decreased the level of IFN-γ secreted by adipocytes and downregulated adi-CM-induced PD-L1 expression in HepG2 cells. PMQ could inhibit HCC progression in obese mice at least in part through down-regulating adipocytes-induced PD-L1 expression via IFN-γ signaling.

LINC00657 activates PD-L1 to promote osteosarcoma metastasis via miR-106a.

Osteosarcoma (OS) cells are one of the primary cancer-related causes of death around the world. Long noncoding RNAs are key for OS progression; however, the detailed molecular mechanism remains unknown. LINC00657, miR-106a, and programmed death ligand-1 (PD-L1) genes expression were detected by reverse transcription-quantitative PCR (RT-qPCR) and Western blotapproaches. Invasion and lymphangiogenesis were studied using transwell invasion assay and lymphatic vessel formation assay, respectively. We used bioinformatic analyses to identify putative targets of LINC00657 and miR-106a. Luciferase activity was measured by dual-luciferase reporter assay. PD-L1 protein levels were examined by flow cytometry experiments. LINC00657 knockdown attenuates cell invasion and tumor growth of MG63 and lymphatic vessel formation. miR-106a directly binds LINC00657 and they regulate each other. Furthermore, miR-106a inhibitor strikingly enhanced lymphatic vessel formation and invasion of shLINC00657 MG63 cells. miR-106a mimic directly targeted and downregulated PD-L1. PD-L1 overexpression largely rescued miR-106a mimic-modulated OS cell metastasis. LINC00657 and PD-L1 were upregulated in clinical OS tumors compared to normal tissues. Lower expression levels of LINC00657 and PD-L1 were closely associated with higher overall survival of patients with OS. Here, we suggest a novel mechanism for LINC00657-regulated metastasis of OS cells by regulating the miR-106a/PD-L1 axis. Our conclusions facilitate the development of therapeutical approaches by targeting LINC00657.