Regulation Of PD-L1 Expression Research Articles

Serum-derived exosomal miR-125a-3p predicts the response to anti-programmed cell death-1/programmed cell death-ligand 1 monotherapy in patients with non-small cell lung cancer

BackgroundVersatile biomarkers for immune checkpoint inhibitors (ICI) efficacy in patients with cancer remain to be identified. Liquid biopsy using serum-derived exosomal microRNAs (miRNAs) are widely investigated as diagnostic and therapeutic outcome predictors in patients with cancer. However, exosomal miRNAs linked to the response to ICI in patients with non-small cell lung cancer (NSCLC) remain elusive thus far. Methods: The value of serum-derived exosomal miRNAs in predicting the effect of anti-programmed cell death-1 (PD-1)/anti-programmed cell death-ligand 1 (PD-L1) monotherapy in 41 patients with advanced NSCLC was assessed. We performed functional analysis of candidate miRNAs using NSCLC cell lines. Results: Exosomal miR-125a-3p was associated with response to treatment with ICI. Exosomal miR-125a-3p was more useful in predicting response to ICI versus tumoral PD-L1 in patients with low PD-L1 expression <50 %). Moreover, high expression of miR-125a-3p was associated with worse progression-free and overall survival. In H1975 and H441 cells, induction of miR-125a-3p regulated PD-L1 expression via suppression of neuregulin 1 (NRG1). Conclusions: Exosomal miR-125a-3p is a potential predictor of response to anti-PD-1/PD-L1 therapy in advanced NSCLC patients with low PD-L1 expression.

SETDB1: A perspective into immune cell function and cancer immunotherapy.

Oncogene SET Domain Bifurcated 1 (SETDB1)/ESET, an H3K9 methyltransferase, was originally discovered over two decades ago; however, its function in the immune response was not first reported until 2011. SETDB1 immune functions include B cell maturation, T cell activity regulation, and immune escape in cancer cells. In B lymphocytes, SETDB1 mediates the transition from pro-B to pre-B cells and represses endogenous retroviruses (ERV) to encourage B cell lineage differentiation and maturation. SETDB1 alters T cell function by methylating IL-2 and IL-17 promoters and mediating T cell lineage commitment and development. In addition, SETDB1 plays a critical role in ERV silencing within a variety of immune cells, which can indirectly weaken the immune response. Although SETDB1 is critical for normal immune cell function, overexpression in cancer cells negatively impacts immune cell fights against cancer through decreased tumour immunogenicity. Within cancer cells, SETDB1 overexpression represses production and infiltration of antitumour immune cells, mediates immune escape through TE and ERV silencing, represses the type I interferon pathway, and interferes in immune checkpoint blockade (ICB) outcomes by regulation of PD-L1 expression and IFN signalling. In this review, we further discuss the immunological mechanisms of SETDB1 in normal and cancerous cells and its implications in cancer immunotherapy.

Expression of nucleus accumbens-1 in colon cancer negatively modulates antitumor immunity.

Nucleus accumbens-1 (NAC-1) is highly expressed in a variety of tumors, including colon cancer, and is closely associated with tumor recurrence, metastasis, and invasion. To determine whether and how NAC-1 affects antitumor immunity in colon cancer. NAC-1-siRNA was transfected into RKO colon cancer cells to knock down NAC expression; tumor cells with or without knockdown of NAC-1 were treated with CD8+ T cells to test their cytocidal effect. The level of the immune checkpoint programmed death receptor-1 ligand (PD-L1) in colon cancer cells with or without knockdown of NAC-1 was analyzed using Quantitative real-time polymerase chain reaction and Western blotting. A double luciferase reporter assay was used to examine the effects of NAC-1 on the transcription of PD-L1. Mice bearing MC-38-OVA colon cancer cells expressing NAC-shRNA or control-shRNA were treated with OT-I mouse CD8+ T cells to determine the tumor response to immunotherapy. Immune cells in the tumor tissues were analyzed using flow cytometry. NAC-1, PD-L1 and CD8+ T cells in colon cancer specimens from patients were examined using immunohistochemistry staining. Knockdown of NAC-1 expression in colon cancer cells significantly enhanced the cytocidal effect of CD8+ T cells in cell culture experiments. The sensitizing effect of NAC-1 knockdown on the antitumor action of cytotoxic CD8+ T cells was recapitulated in a colon cancer xenograft animal model. Furthermore, knockdown of NAC-1 in colon cancer cells decreased the expression of PD-L1 at both the mRNA and protein levels, and this effect could be rescued by transfection of an RNAi-resistant NAC-1 expression plasmid. In a reporter gene assay, transient expression of NAC-1 in colon cancer cells increased the promoter activity of PD-L1, indicating that NAC-1 regulates PD-L1 expression at the transcriptional level. In addition, depletion of tumoral NAC-1 increased the number of CD8+ T cells but decreased the number of suppressive myeloid-derived suppressor cells and regulatory T cells. Tumor expression of NAC-1 is a negative determinant of immunotherapy.

Dickkopf-1 drives tumor immune evasion by inducing PD-L1 expression in hepatocellular carcinoma

Understanding the mechanisms regulating PD-L1 expression in hepatocellular carcinoma (HCC) is important to improve the response rate to PD-1/PD-L1 blockade therapy. Here, we show that DKK1 expression is positively associated with PD-L1 expression and inversely correlated with CD8+ T cell infiltration in human HCC tumor specimens. In a subcutaneous xenograft tumor model, overexpression of DKK1 significantly promotes tumor growth, tumoral PD-L1 expression, but reduces tumoral CD8+ T cell infiltration; whereas knockdown of DKK1 has opposite effects. Moreover, enforced expression of DKK1 dramatically promotes PD-L1 expression, Akt activation, β-catenin phosphorylation and total protein expression in HCC cells. By contrast, knockdown of DKK1 inhibits all, relative to controls. In addition, CKAP4 depletion, Akt inhibition, or β-catenin depletion remarkably abrogates DKK1 overexpression-induced transcriptional expression of PD-L1 in HCC cells. Reconstituted expression of the active Akt1 largely increased PD-L1 transcriptional expression in HCC cells. Similarly, expression of WT β-catenin, but not the phosphorylation-defective β-catenin S552A mutant, significantly promotes PD-L1 expression. Correlation analysis of human HCC tumor specimens further revealed that DKK1 and PD-L1 expression were positively correlated with p-β-catenin expression. Together, our findings revealed that DKK1 promotes PD-L1 expression through the activation of Akt/β-catenin signaling, providing a potential strategy to enhance the clinical efficacy of PD-1/PD-L1 blockade therapy in HCC patients.

HSF1 is involved in immunotherapeutic response through regulating APOJ/STAT3-mediated PD-L1 expression in hepatocellular carcinoma

ABSTRACT Hepatocellular cancer (HCC) is a serious illness with high prevalence and mortality throughout the whole world. For advanced HCC, immunotherapy is somewhat impactful and encouraging. Nevertheless, a substantial proportion of patients with advanced HCC are still unable to achieve a durable response, owing to heterogeneity from clonal variability and differential expression of the PD-1/PD-L1 axis. Recently, heat shock factor 1 (HSF1) is recognized as an important component of tumor immunotherapeutic response as well as related to PD-L1 expression in cancer. However, the mechanism of HSF1 regulating PD-L1 in cancer, especially in HCC, is still not fully clear. In this study, we observed the significantly positive correlation between HSF1 expression and PD-L1 expression in HCC samples; meanwhile combination expressions of HSF1 and PD-L1 served as the signature for predicting prognosis of patients with HCC. Mechanistically, HSF1 upregulated PD-L1 expression by inducing APOJ expression and activating STAT3 signaling pathway in HCC. In addition, we explored further the potential values of targeting the HSF1-APOJ-STAT3 axis against CD8+ T cells-mediated cancer cells cytotoxicity. These findings unveiled the important involvement of HSF1 in regulating PD-L1 expression in HCC as well as provided a novel invention component for improving the clinical response rate and efficacy of PD-1/PD-L1 blockade.

SPOP mutations promote tumor immune escape in endometrial cancer via the IRF1-PD-L1 axis.

Blockade of programmed cell death 1 (PD-1)/programmed cell death 1 ligand (PD-L1) has evolved into one of the most promising immunotherapy strategies for cancer patients. Tumor cells frequently overexpress PD-L1 to evade T cell-mediated immune surveillance. However, the specific genetic alterations that drive aberrant overexpression of PD-L1 in cancer cells remain poorly understood. The gene encoding the E3 ubiquitin ligase substrate-binding adaptor SPOP is frequently mutated in endometrial cancer (EC). Here, we report that SPOP negatively regulates PD-L1 expression at the transcriptional level. Wild-type SPOP binds to IRF1, a primary transcription factor responsible for the inducible expression of PD-L1, and subsequently triggers its ubiquitin- proteasomal degradation to suppress IRF1-mediated transcriptional upregulation of PD-L1. In contrast, EC-associated SPOP mutants lose their capacity to degrade IRF1 but stabilize IRF1, and upregulate PD-L1 expression. EC-associated SPOP mutations accelerate xenograft tumor growth partially by increasing IRF1 and PD-L1 expression. Together, we identify SPOP as a negative regulator of the IRF1-PD-L1 axis and characterize the critical roles of IRF1 and PD-L1 in SPOP mutation-driven tumor immune evasion in EC.

Formin protein DIAPH1 positively regulates PD-L1 expression and predicts the therapeutic response to anti-PD-1/PD-L1 immunotherapy

Formins are evolutionarily conserved genes and profoundly affect cancer progression. This study aims to explore the expressions, prognostic values, and immunological correlations of Formins in cancer. Specific Formins were dysregulated and immuno-biologically correlated in breast cancer (BRCA). Formins showed different expression patterns, namely some were enriched in immune cells while some were enriched in tumor cells. Among all Formins, DIAPH1 was enriched in tumor cells and associated with an inflamed tumor microenvironment (TME). DIAPH1 functioned as an oncogene in BRCA and mediated TGF-β1-induced epithelial-mesenchymal transformation (EMT) and PD-L1 expression. Moreover, DIAPH1 was overexpressed in most cancers and functioned as a novel pan-cancer immuno-marker, which could predict the response to anti-PD-1/PD-L1 immunotherapy. Overall, DIAPH1 functions as an oncogene and is immunologically correlated, which could be utilized as an alternative biomarker for predicting the immunotherapeutic response.

Integrated Chemical Characterization, Network Pharmacology and Transcriptomics to Explore the Mechanism of Sesquiterpenoids Isolated from Gynura divaricata (L.) DC. against Chronic Myelogenous Leukemia.

Chronic myelogenous leukemia (CML) is a serious threat to human health, while drugs for CML are limited. Herbal medicines with structural diversity, low toxicity and low drug resistance are always the most important source for drug discoveries. Gynura divaricata (L.) DC. is a well-known herbal medicine whose non-alkaline ingredients (GD-NAIs) were isolated. The GD-NAIs demonstrated potential anti-CML activity in our preliminary screening tests. However, the chemical components and underlying mechanism are still unknown. In this study, GD-NAIs were tentatively characterized using UHPLC-HRMS combined with molecular networking, which were composed of 75 sesquiterpenoids. Then, the anti-CML activities of GD-NAIs were evaluated and demonstrated significant suppression of proliferation and promotion of apoptosis in K562 cells. Furthermore, the mechanism of GD-NAIs against CML were elucidated using network pharmacology combined with RNA sequencing. Four sesquiterpenoids would be the main active ingredients of GD-NAIs against CML, which could regulate PD-L1 expression and the PD-1 checkpoint pathway in cancer, PI3K/AKT, JAK/STAT, TGF-β, estrogen, Notch and Wnt signaling pathways. In conclusion, our study reveals the composition of GD-NAIs, confirms its anti-CML activity and elucidates their underlying mechanism, which is a potential countermeasure for the treatment of CML.

447 Development of the combination therapy of anti-PD1 antibody with PAI-1 inhibitors in advanced melanoma patients

Plasminogen activating inhibitor-1 (PAI-1) is associated with poor clinical outcomes in cancer patients, and elevated levels of PAI-1 in both tissue and serum are correlated with poor response to therapy in various cancers, including skin cancer. PAI-1 is reported as a key regulator of PD-L1 expression on melanoma cells through endocytosis, leading to abrogating the efficacy of anti-PD1 antibodies (Abs). These findings suggested that PAI-1 expression might predict the efficacy of anti-PD1 Abs. From the above findings, at first, we evaluated the expression and production of PAI-1 in melanoma patients who treated with anti-PD1 Abs, and then, we investigated the immunomodulatory effects of PAI-1 on tumor-associated macrophages in vitro.

Elevation of spermine remodels immunosuppressive microenvironment through driving the modification of PD-L1 in hepatocellular carcinoma

BackgroundSpermine is frequently elevated in tumor tissues and body fluids of cancer patients and is critical for cancer cell proliferation, migration and invasion. However, the immune functions of spermine in hepatocellular carcinoma progression remains unknown. In the present study, we aimed to elucidate immunosuppressive role of spermine in hepatocellular carcinoma and to explore the underlying mechanism.MethodsWhole-blood spermine concentration was measured using HPLC. Human primary HCC tissues were collected to examine the expression of CaSR, p-Akt, β-catenin, STT3A, PD-L1, and CD8. Mouse model of tumorigenesis and lung metastasis were established to evaluate the effects of spermine on hepatocellular carcinoma. Western blotting, immunofluorescence, real time PCR, digital Ca2+ imaging, and chromatin immunoprecipitation assay were used to investigate the underlying mechanisms by which spermine regulates PD-L1 expression and glycosylation in hepatocellular carcinoma cells.ResultsBlood spermine concentration in the HCC patient group was significantly higher than that in the normal population group. Spermine could facilitate tumor progression through inducing PD-L1 expression and decreasing the CD8+ T cell infiltration in HCC. Mechanistically, spermine activates calcium-sensing receptor (CaSR) to trigger Ca2+ entry and thereby promote Akt-dependent β-catenin stabilization and nuclear translocation. Nuclear β-catenin induced by spermine then activates transcriptional expression of PD-L1 and N-glycosyltransferase STT3A, while STT3A in turn increases the stability of PD-L1 through inducing PD-L1 protein N-glycosylation in HCC cells.ConclusionsThis study reveals the crucial function of spermine in establishing immune privilege by increasing the expression and N-glycosylation of PD-L1, providing a potential strategy for the treatment of hepatocellular carcinoma.7ng36xqHia8rCQn62itWxRVideo

E3 Ubiquitin Ligase RNF125 Suppresses Immune Escape in Head and Neck Squamous Cell Carcinoma by Regulating PD-L1 Expression.

Head and neck squamous cell carcinoma (HNSCC) is a lethal malignancy. Given the essential roles of E3 ligases in cancer immunotherapies, this paper explored the effect of E3 ubiquitin ligase ring finger protein 125 (RNF125) on immune escape in HNSCC. After delivering overexpressed (oe)-RNF125, interferon-gamma, or oe-programmed death-ligand 1 (PD-L1) into HNSCC cells and cell culture with carboxyfluorescein succinimidyl ester-labeled CD8+ T cells, RNF125 and PD-L1 levels were determined via RT-qPCR and Western blot, with HNSCC cell behaviors assessed via colony formation assay, Transwell assays and flow cytometry, and inflammatory factors measured via ELISA. PD-L1 ubiquitination level and PD-L1's interaction with RNF125 were analyzed via co-immunoprecipitation. The in vivo action of RNF125 on HNSCC was validated via nude mouse tumorigenicity assay. Briefly, RNF125 was weakly expressed in HNSCC cells. RNF125 overexpression inhibited immune escape of HNSCC cells, evidenced by decreased TSCCA cell proliferation, migration, and invasion, increased CD8+ T cell proliferation, and elevated IL-2 and TNF-α levels. RNF125 downregulated PD-L1 in TSCCA cells and facilitated PD-L1 ubiquitinational degradation. PD-L1 overexpression partially abated RNF125-mediated suppression on TSCCA cell immune escape. Moreover, RNF125 suppressed tumorigenesis and tumor growth in vivo. Overall, RNF125 promoted PD-L1 ubiquitinational degradation, hence inhibiting immune escape in HNSCC.

Identification of CKS1B as a prognostic indicator and a predictive marker for immunotherapy in pancreatic cancer.

As a regulatory subunit of cyclin kinase, CKS1B promotes cancer development and is associated with poor prognosis in multiple cancer patients. However, the intrinsic role of CKS1B in pancreatic cancer remains elusive. In our research, CKS1B expression in pancreatic tumor tissue was higher than that in normal tissue by TCGA, Oncomine and CPTAC databases analysis. Similar result was verified in our center tissues by qRT-PCR. CKS1B expression was closely relevant to histologic grading, prognosis, and TMB. GSEA showed that CKS1B mainly participated in the regulation of autophagy and T cell receptor signaling pathway. Furthermore, CIBERSORT analysis showed that there was a strong correlation between CKS1B expression and tumor immune cells infiltration. Drug sensitivity analysis showed that patients with high CKS1B expression appeared to be more sensitive to gemcitabine, 5-fluorouracil, and paclitaxel. We then investigated cell viability and migratory ability by CCK8 and transwell assay, respectively. Results indicated that CKS1B knockdown by short hairpin RNA significantly reduced pancreatic cancer cell viability and invasion via regulating PD-L1 expression. In conclusion, our research further demonstrates the role of CKS1B in pancreatic cancer and the signaling pathways involved. The association of CKS1B with immune infiltration and immune checkpoint may provide a new direction for immunotherapy of pancreatic cancer.

Circ_0058058 Drives the Malignant Phenotypes and Immune Evasion of Pancreatic Cancer by the MicroRNA-557-Dependent Regulation of PDL1.

Pancreatic cancer (PC) is one of the most deadly malignancies in the world. Recently, circular RNAs play crucial roles in PC progression. However, the functions of circ_0058058 in PC are barely known. The expression of circ_0058058, microRNA-557-5p (miR-557), and programmed cell death receptor ligand 1 (PDL1) was detected by quantitative real-time polymerase chain reaction. Functional experiments were implemented to disclose the effect of circ_0058058 deficiency on PC cell proliferation, apoptosis, invasion, angiogenesis, and immune escape. The binding relationship between miR-557 and circ_0058058 or PDL1 was identified by dual-luciferase reporter assay and RNA immunoprecipitation assay. In vivo assay was used to disclose the impact of circ_0058058 silencing on tumor formation in vivo. Circ_0058058 was highly expressed in PC tissues and cell lines. Knockdown of circ_0058058 repressed cell proliferation, invasion, angiogenesis, and immune escape while contributed to apoptosis in PC cells. Mechanically, circ_0058058 worked as a molecular sponge of miR-557 to regulate PDL1 expression. Moreover, circ_0058058 showed a promotional effect on tumor growth in vivo. Our findings suggested that circ_0058058 served as miR-557 sponge to upregulate PDL1, thereby triggering PC proliferation, invasion, angiogenesis, and immune escape.

Visible- and near-infrared hyperspectral imaging for the quantitative analysis of PD-L1+ cells in human lymphomas: Comparison with fluorescent multiplex immunohistochemistry

IntroductionWe analyzed the expression of PD-L1 in human lymphomas using hyperspectral imaging (HSI) compared to visual assessment (VA) and conventional digital image analysis (DIA) to strengthen further the value of HSI as a tool for the evaluation of brightfield-based immunohistochemistry (IHC). In addition, fluorescent multiplex immunohistochemistry (mIHC) was used as a second detection method to analyze the impact of a different detection method. Material and methods18 cases (6 follicular lymphomas and 12 diffuse large B-cell lymphomas) were stained for PD-L1 by IHC and for PD-L1, CD3, and CD8 by fluorescent mIHC. The percentage of positively stained cells was evaluated with VA, HSI, and DIA for IHC and VA and DIA for mIHC. Results were compared between the different methods of detection and analysis. ResultsAn overall high concordance was found between VA, HSI, and DIA in IHC (Cohens Kappa = 0.810VA/HSI, 0.710 VA/DIA, and 0.516 HSI/DIA) and for VAmIHCversus DIAmIHC (Cohens Kappa = 0.894). Comparing IHC and mIHC general agreement differed depending on the methods compared but reached at most a moderate agreement (Coheńs Kappa between 0.250 and 0.483). This is reflected by the significantly higher percentage of PD-L1+ cells found with mIHC (pFriedman = 0.014). ConclusionOur study shows a good concordance for the different analysis methods. Compared to VA and DIA, HSI proved to be a reliable tool for assessing IHC. Understanding the regulation of PD-L1 expression will further enlighten the role of PD-L1 as a biomarker. Therefore it is necessary to develop an instrument, such as HSI, which can offer a reliable and objective evaluation of PD-L1 expression.

MCT4/Lactate Promotes PD-L1 Glycosylation in Triple-Negative Breast Cancer Cells.

Triple-negative breast cancer (TNBC) has the highest percentage of lymphocytic infiltration among breast cancer subtypes, and TNBC patients may benefit from anti-PD-1/PD-L1 immunotherapy. However, some cases whether the immune checkpoint blockade (ICB) shows low targeting efficiency have occurred and effective synergistic targets need to be found, which inspired our exploration of the co-expression analysis of MCT4 (SLC16A3) and PD-L1 (CD274) and their potential regulatory mechanisms. After bioinformatic analysis of the relationship between MCT4 and PD-L1, we validated their positive co-expression relationship in triple-negative breast cancer through multiple immunohistochemical staining (mIHC), CRISPR/Cas9, and lentiviral transduction for MCT4 knockout (sgMCT4/231 KO) or overexpression (pEGFP-N1-MCT4/231). We examined the effect of lactate treatment on PD-L1 expression in triple-negative breast cancer cells by qRT-PCR and Western blot. Combined with our results, we found that MCT4 positively regulated PD-L1 expression through discharging lactate and stabilized PD-L1 through promoting its glycosylation by the classic WNT pathway in MDA-MB-231 cells. More importantly, the high co-expression of MCT4 and PD-L1 appears to predict more effective targets for treating TNBC, which would improve immune checkpoint therapy for TNBC.

Qingfei Jiedu decoction inhibits PD-L1 expression in lung adenocarcinoma based on network pharmacology analysis, molecular docking and experimental verification.

Objective: We aim at investigating the molecular mechanisms through which the Qingfei Jiedu decoction (QFJDD) regulates PD-L1 expression in lung adenocarcinoma (LUAD). Methods: Bioactive compounds and targets of QFJDD were screened from TCMSP, BATMAN-TCM, and literature. Then, GeneCard, OMIM, PharmGKB, Therapeutic Target, and DrugBank databases were used to identify LUAD-related genes. The protein-protein interaction (PPI) network was constructed using overlapping targets of bioactive compounds in LUAD with the Cytoscape software and STRING database. The potential functions and pathways in which the hub genes were enriched by GO, KEGG, and DAVID pathway analyses. Molecular docking of bioactive compounds and key genes was executed via AutoDock Vina. Qualitative and quantitative analyses of QFJDD were performed using UPLC-Q-TOF-MS and UPLC. Expressions of key genes were determined by qRT-PCR, immunoreactivity score (IRS) of PD-L1 was assessed by immunohistochemistry (IHC), while the CD8+PD-1+T% derived from spleen tissues of Lewis lung cancer (LLC) bearing-mice was calculated using flow cytometry (FCM). Results: A total of 53 bioactive compounds and 288 targets of QFJDD as well as 8151 LUAD associated genes were obtained. Further, six bioactive compounds, including quercetin, luteolin, kaempferol, wogonin, baicalein, and acacetin, and 22 hub genes were identified. The GO analysis showed that the hub genes were mainly enriched in DNA or RNA transcription. KEGG and DAVID pathway analyses revealed that 20 hub genes were primarily enriched in virus, cancer, immune, endocrine, and cardiovascular pathways. The EGFR, JUN, RELA, HIF1A, NFKBIA, AKT1, MAPK1, and MAPK14 hub genes were identified as key genes in PD-L1 expression and PD-1 checkpoint pathway. Moreover, ideal affinity and regions were identified between core compounds and key genes. Notably, QFJDD downregulated EGFR, JUN, RELA, HIF1A, NFKBIA, and CD274 expressions (p < 0.05), while it upregulated AKT1 and MAPK1 (p < 0.05) levels in A549 cells. The PD-L1 IRS of LLC tissue in the QFJDD high dose (Hd) group was lower than model group (p < 0.01). CD8+PD-1+T% was higher in the QFJDD Hd group than in normal and model groups (p < 0.05). Conclusion: QFJDD downregulates PD-L1 expression and increases CD8+PD-1+T% via regulating HIF-1, EGFR, JUN and NFκB signaling pathways. Therefore, QFJDD is a potential treatment option for LUAD.

Phosphofructokinase 1 platelet isoform induces PD-L1 expression to promote glioblastoma immune evasion.

Overexpression of PD-L1 is observed in many types of human cancer, including glioblastoma (GBM) and contributes to tumor immune evasion. In addition, GBM shows highly-activated aerobic glycolysis due to overexpression of phosphofructokinase 1 platelet isoform (PFKP), which the key enzyme in the glycolysis. However, it remains unclear whether the metabolic enzyme PFKP plays a role in the regulation of PD-L1 expression and GBM immune evasion. We aimed to investigate the non-metabolic role of PFKP in PD-L1 expression-induced GBM immune evasion. The mechanisms of PFKP-induced PD-L1 expression were studied by several experiments, including real-time PCR, immunoblot analysis, and ATP production. The coculture experiments using GBM cell and T cells were performed to evaluate the effect of PFKP on T cell activation. The clinical relationship between PFKP and PD-L1 was analyzed in The Cancer Genome Atlas (TCGA) database and in human GBM specimens. We showed that PFKP promotes EGFR activation-induced PD-L1 expression in human GBM cells. Importantly, we demonstrated that EGFR-phosphorylated PFKP Y64 plays an important role in AKT-mediated β-catenin transactivation and subsequent PD-L1 transcriptional expression, thereby enhancing the GBM immune evasion. In addition, based on our findings, the levels of PFKP Y64 phosphorylation are positively correlated with PD-L1 expression in human GBM specimens, highlighting the clinical significance of PFKP Y64 phosphorylation in the GBM immune evasion. These findings provide new mechanistic insight into the regulation of PD-L1 expression by a non-metabolic function of PFKP on tumor cells.

Immunorthodontics: PD-L1, a Novel Immunomodulator in Cementoblasts, Is Regulated by HIF-1α under Hypoxia.

Recent studies have revealed that hypoxia alters the PD-L1 expression in periodontal cells. HIF-1α is a key regulator for PD-L1. As hypoxia presents a hallmark of an orthodontically induced microenvironment, hypoxic stimulation of PD-L1 expression may play vital roles in immunorthodontics and orthodontically induced inflammatory root resorption (OIIRR). This study aims to investigate the hypoxic regulation of PD-L1 in cementoblasts, and its interaction with hypoxia-induced HIF-1α expression. The cementoblast (OCCM-30) cells (M. Somerman, NIH, NIDCR, Bethesda, Maryland) were cultured in the presence and absence of cobalt (II) chloride (CoCl2). Protein expression of PD-L1 and HIF-1α as well as their gene expression were evaluated by Western blotting and RT-qPCR. Immunofluorescence was applied to visualize the localization of the proteins within cells. The HIF-1α inhibitor (HY-111387, MedChemExpress) was added, and CRISPR/Cas9 plasmid targeting HIF-1α was transferred for further investigation by flow cytometry analysis. Under hypoxic conditions, cementoblasts undergo an up-regulation of PD-L1 expression at protein and mRNA levels. Silencing of HIF-1α using CRISPR/Cas9 indicated a major positive correlation with HIF-1α in regulating PD-L1 expression. Taken together, these findings show the influence of hypoxia on PD-L1 expression is modulated in a HIF-1α dependent manner. The HIF-1α/PD-L1 pathway may play a role in the immune response of cementoblasts. Thus, combined HIF-1α/PD-L1 inhibition could be of possible therapeutic relevance for OIIRR prevention.

Ubiquitin ligase RNF125 targets PD-L1 for ubiquitination and degradation.

As a critical immune checkpoint molecule, PD-L1 is expressed at significantly higher levels in multiple neoplastic tissues compared to normal ones. PD-L1/PD-1 axis is a critical target for tumor immunotherapy, blocking the PD-L1/PD-1 axis is recognized and has achieved unprecedented success in clinical applications. However, the clinical efficacy of therapies targeting the PD-1/PD-L1 pathway remains limited, emphasizing the need for the mechanistic elucidation of PD-1/PD-L1 expression. In this study, we found that RNF125 interacted with PD-L1 and regulated PD-L1 protein expression. Mechanistically, RNF125 promoted K48-linked polyubiquitination of PD-L1 and mediated its degradation. Notably, MC-38 and H22 cell lines with RNF125 knockout, transplanted in C57BL/6 mice, exhibited a higher PD-L1 level and faster tumor growth than their parental cell lines. In contrast, overexpression of RNF125 in MC-38 and H22 cells had the opposite effect, resulting in lower PD-L1 levels and delayed tumor growth compared with parental cell lines. In addition, immunohistochemical analysis of MC-38 tumors with RNF125 overexpression showed significantly increased infiltration of CD4+, CD8+ T cells and macrophages. Consistent with these findings, analyses using The Cancer Genome Atlas (TCGA) public database revealed a positive correlation of RNF125 expression with CD4+, CD8+ T cell and macrophage tumor infiltration. Moreover, RNF125 expression was significantly downregulated in several human cancer tissues, and was negatively correlated with the clinical stage of these tumors, and patients with higher RNF125 expression had better clinical outcomes. Our findings identify a novel mechanism for regulating PD-L1 expression and may provide a new strategy to increase the efficacy of immunotherapy.

HER-3 molecular classification, expression of PD-L1 and clinical importance in breast cancer.

Receptors of the large HER family play an important role in breast cancer, which is undergoing a gradual development in connection with biological development, both in the field of diagnostics and therapy. Dimerization of HER-2 with other HER members, such as HER-3, is the biggest driver of tumor cell growth and survival. Numerous studies show that HER-3 gene overexpression correlates with poor prognosis. However, other studies have shown HER-3 overexpression to be a positive prognostic factor. HER-3 may confer resistance to certain EGFR or HER-2 receptor therapeutics. An interesting fact, however, is that HER-3 expression can serve as a marker in immunotherapy for triple-negative breast cancer (TNBC). It is thought to be involved not only in cell survival and proliferation, but also in the regulation of PD-L1 expression. In breast cancer, PD-L1 expression is heterogeneous and is generally associated with the presence of tumor‑infiltrating lymphocytes and a number of factors with poor prognosis such as young age, hormone receptor negativity, and high HER-2 expression and proliferation index. Our results showed amplification of HER-3 (CERB3) in 2 out of a sample of 20 patients with TNBC, and 13 of 20 HER-2‑positive patients. PD-L1 expression was demonstrated in 3 out of 13 HER-3‑positive patients and 2 out of 2 HER-3‑positive TNBC patients. There was a strong correlation between positive HER-3 and PD-L1 TNBC expression (p = Keywords: breast cancer, HER family, overexpression, HER-3, HER-2, PD-L1, TNBC.