RESUMO
Lymphocyte-activation gene 3 (LAG-3) is an immune inhibitory receptor, with major histocompatibility complex class II (MHC-II) as a canonical ligand. However, it remains controversial whether MHC-II is solely responsible for the inhibitory function of LAG-3. Here, we demonstrate that fibrinogen-like protein 1 (FGL1), a liver-secreted protein, is a major LAG-3 functional ligand independent from MHC-II. FGL1 inhibits antigen-specific T cell activation, and ablation of FGL1 in mice promotes T cell immunity. Blockade of the FGL1-LAG-3 interaction by monoclonal antibodies stimulates tumor immunity and is therapeutic against established mouse tumors in a receptor-ligand inter-dependent manner. FGL1 is highly produced by human cancer cells, and elevated FGL1 in the plasma of cancer patients is associated with a poor prognosis and resistance to anti-PD-1/B7-H1 therapy. Our findings reveal an immune evasion mechanism and have implications for the design of cancer immunotherapy.
Assuntos
Antígenos CD/metabolismo , Proteínas de Neoplasias/metabolismo , Proteínas de Neoplasias/fisiologia , Animais , Antígenos CD/imunologia , Linhagem Celular , Fibrinogênio/imunologia , Fibrinogênio/metabolismo , Genes MHC da Classe II/genética , Genes MHC da Classe II/imunologia , Antígenos de Histocompatibilidade Classe II/genética , Antígenos de Histocompatibilidade Classe II/imunologia , Antígenos de Histocompatibilidade Classe II/metabolismo , Humanos , Imunoterapia , Ligantes , Fígado/metabolismo , Ativação Linfocitária/imunologia , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Knockout , Proteínas de Neoplasias/genética , Neoplasias/imunologia , Linfócitos T Citotóxicos/imunologia , Proteína do Gene 3 de Ativação de LinfócitosRESUMO
Lymphocyte activation gene-3 (LAG-3) is a potent inhibitory co-receptor; yet, its functional ligand remains elusive, with distinct potential ligands identified. Here, we investigated the relative contribution of potential ligands, stable peptide-MHC class II complexes (pMHCII) and fibrinogen-like protein 1 (FGL1), to LAG-3 activity in vitro and in vivo. Binding of LAG-3 to stable pMHCII but not to FGL1 induced T cell suppression in vitro. Consistently, LAG-3 mutants lacking FGL1-binding capacity but not those lacking stable pMHCII-binding capacity retained suppressive activity in vitro. Accordingly, targeted disruption of stable pMHCII- but not FGL1-binding capacity of LAG-3 in NOD mice recapitulated diabetes exacerbation by LAG-3 deficiency. Additionally, the loss of stable pMHCII-binding capacity of LAG-3 augmented anti-cancer immunity comparably with LAG-3 deficiency in C57BL/6 mice. These results identify stable pMHCII as a functional ligand of LAG-3 both in autoimmunity and anti-cancer immunity. Thus, stable pMHCII-LAG-3 interaction is a potential therapeutic target in human diseases.
Assuntos
Antígenos CD , Autoimunidade , Antígenos de Histocompatibilidade Classe II , Neoplasias , Linfócitos T , Animais , Antígenos CD/metabolismo , Antígenos de Histocompatibilidade Classe II/metabolismo , Ligantes , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Endogâmicos NOD , Neoplasias/imunologia , Peptídeos/metabolismo , Linfócitos T/imunologia , Proteína do Gene 3 de Ativação de LinfócitosRESUMO
Lymphocyte activation gene 3 protein (LAG3) is an immune checkpoint receptor that is highly upregulated on exhausted T cells in the tumor microenvironment. LAG3 transmits inhibitory signals to T cells upon binding to MHC class II and other ligands, rendering T cells dysfunctional. Consequently, LAG3 is a major target for cancer immunotherapy with many anti-LAG3 monoclonal antibodies (mAbs) that block LAG3 inhibitory activity in clinical trials. In this review, we examine the molecular basis for LAG3 function in light of recently determined crystal and cryoEM structures of this inhibitory receptor. We review what is known about LAG3 interactions with MHC class II, its canonical ligand, and the newly discovered ligands FGL1 and the T cell receptor (TCR)-CD3 complex, including current controversies over the relative importance of these ligands. We then address the development and mechanisms of action of anti-LAG3 mAbs in clinical trials for cancer immunotherapy. We discuss new strategies to therapeutically target LAG3 using mAbs that not only block the LAG3-MHC class II interaction, but also LAG3 interactions with FGL1 or TCR-CD3, or that disrupt LAG3 dimerization. Finally, we assess the possibility of developing mAbs that enhance, rather than block, LAG3 inhibitory activity as treatments for autoimmune diseases.
Assuntos
Antígenos CD , Imunoterapia , Proteína do Gene 3 de Ativação de Linfócitos , Neoplasias , Animais , Humanos , Antígenos CD/imunologia , Antígenos CD/metabolismo , Antígenos CD/química , Imunoterapia/métodos , Proteína do Gene 3 de Ativação de Linfócitos/imunologia , Neoplasias/terapia , Neoplasias/imunologia , Neoplasias/metabolismo , Receptores de Antígenos de Linfócitos T/imunologia , Receptores de Antígenos de Linfócitos T/metabolismo , Receptores de Antígenos de Linfócitos T/químicaRESUMO
Fibrinogen-like protein-1 (FGL1) is confirmed a major ligand of lymphocyte activation gene-3 which could inhibit antigen-mediated T-cell response and evade immune supervision. Although hepatocytes secrete large amounts of FGL1, its high expression also be detected in solid tumors such as lung cancer, leading to a poor efficacy of immune checkpoint inhibitors therapy. Here we reported that FGL1 was overexpressed in lung adenocarcinoma (LUAD) but not in lung squamous cell carcinoma. However, FGL1 in tissue and plasma can only distinguish LUAD patients from healthy donors and cannot correlate with clinical Tumor Node Metastasis (TNM) stage. Using lung cancer cell lines, we confirmed that FGL1 can be detected on extracellular vesicles (EVs) and we established a method using flow cytometry to detect FGL1 on the surface of EVs, which revealed that FGL1 could be secreted via EVs. Both animal model and clinical samples proved that plasma FGL1 in EVs would increase when the tumor was loaded. The level of FGL1 in plasma EVs was correlated with clinical TNM stage and tumor size, and a higher level indicated non-responsiveness to anti-programmed cell death ligand 1 (anti-PD-L1) immunotherapy. Its effect on tumor progression and immune evasion may be achieved by impairing the killing and proliferating capacities of CD8+ T cells. Our result demonstrates that FGL1 levels in plasma EVs, but not total plasma FGL1, could be a promising biomarker that plays an important role in predicting anti-PD-L1 immune therapy in LUAD and suggests a new strategy in LUAD immunotherapy.
Assuntos
Adenocarcinoma de Pulmão , Carcinoma Pulmonar de Células não Pequenas , Vesículas Extracelulares , Neoplasias Pulmonares , Animais , Humanos , Ligantes , Neoplasias Pulmonares/patologia , Carcinoma Pulmonar de Células não Pequenas/patologia , Vesículas Extracelulares/metabolismo , Antígeno B7-H1 , Fibrinogênio/metabolismoRESUMO
BACKGROUND: While radiation therapy remains pivotal in esophageal squamous cell carcinoma (ESCC) treatment, the perplexing phenomenon of post-radiation metastasis presents a formidable clinical challenge. This study investigates the role of fibrinogen-like protein 1 (FGL1) in driving ESCC metastasis following radiation exposure. METHODS: FGL1 expression in post-radiation ESCC cells was meticulously examined using qRT-PCR, western blotting, and immunofluorescence. The impact of FGL1 on ESCC cell invasion and migration was assessed through Transwell and wound healing assays. In vivo, the metastatic potential of ESCC in response to FGL1 was scrutinized using nude mice models. Comprehensive RNA sequencing and functional experiments elucidated the intricate mechanism associated with FGL1. RESULTS: Radiation induced upregulation of FGL1 in ESCC cells through FOXO4, intensifying ESCC cell invasion and migration. Targeted knockdown of FGL1 effectively alleviated these characteristics both in vitro and in vivo. FGL1 depletion concurrently suppressed IMPDH1 expression. Rescue experiments underscored that IMPDH1 knockdown robustly reversed the pro-invasive effects induced by FGL1 in ESCC cells. ESCC tissues exhibited heightened IMPDH1 mRNA levels, demonstrating a correlation with patient survival. CONCLUSIONS: Radiation-induced upregulation of FGL1 propels ESCC metastasis through IMPDH1, proposing a potential therapeutic target to mitigate post-radiotherapy metastasis in ESCC patients.
Assuntos
Movimento Celular , Neoplasias Esofágicas , Carcinoma de Células Escamosas do Esôfago , Regulação para Cima , Humanos , Carcinoma de Células Escamosas do Esôfago/genética , Carcinoma de Células Escamosas do Esôfago/patologia , Carcinoma de Células Escamosas do Esôfago/radioterapia , Carcinoma de Células Escamosas do Esôfago/metabolismo , Animais , Neoplasias Esofágicas/genética , Neoplasias Esofágicas/patologia , Neoplasias Esofágicas/radioterapia , Neoplasias Esofágicas/metabolismo , Camundongos , Linhagem Celular Tumoral , Movimento Celular/genética , Camundongos Nus , Regulação Neoplásica da Expressão Gênica/efeitos da radiação , Metástase Neoplásica , Invasividade Neoplásica/genética , Feminino , MasculinoRESUMO
Fibrinogen-like protein 1 (FGL1) is a potential novel immune checkpoint target for malignant tumor diagnosis and therapy. Accurate detection of FGL1 levels in tumors via noninvasive PET imaging might be beneficial for managing the disease. To achieve this, multiple FGL1-targeting peptides (FGLP) were designed, and a promising candidate, 68Ga-NOTA-FGLP2, was identified through a high-throughput screening approach using microPET imaging of 68Ga-labeled peptides. Subsequent in vitro cell experiments showed that uptake values of 68Ga-NOTA-FGLP2 in FGL1 positive Huh7 tumor cells were significantly higher than those in FGL1 negative U87 MG tumor cells. Further microPET imaging showed that the Huh7 xenografts were clearly visualized with a favorable contrast. ROI analysis showed that the uptake values of the tracer in Huh7 xenografts were 2.63 ± 0.07% ID/g at 30 min p.i.. After treatment with an excess of unlabeled FGLP2, the tumor uptake significantly decreased to 0.54 ± 0.05% ID/g at 30 min p.i.. Moreover, the uptake in U87 MG xenografts was 0.44 ± 0.06% ID/g at the same time point. The tracer was excreted mainly through the renal system. 18F-FDG PET imaging was also performed in mice bearing Huh7 and U87 MG xenografts, respectively. However, there was no significant difference in the uptake between the tumors with different FGL1 expressions. Preclinical data indicated that 68Ga-NOTA-FGLP2 might be a suitable radiotracer for in vivo noninvasive visualization of tumors with abundant expression of FGL1. Further investigation of 68Ga-NOTA-FGLP2 for tumor diagnosis and therapy is undergoing.
Assuntos
Radioisótopos de Gálio , Tomografia por Emissão de Pósitrons , Compostos Radiofarmacêuticos , Animais , Humanos , Camundongos , Tomografia por Emissão de Pósitrons/métodos , Linhagem Celular Tumoral , Compostos Radiofarmacêuticos/farmacocinética , Camundongos Nus , Distribuição Tecidual , Peptídeos/química , Camundongos Endogâmicos BALB C , Feminino , Compostos Heterocíclicos com 1 Anel/químicaRESUMO
Lymphocyte activation gene 3 (LAG3), an immune checkpoint molecule expressed on activated T cells, functions as a negative regulator of immune responses. Persistent antigen exposure in the tumor microenvironment results in sustained LAG3 expression on T cells, contributing to T cell dysfunction. Fibrinogen-like protein 1 (FGL1) has been identified as a major ligand of LAG3, and FGL1/LAG3 interaction forms a novel immune checkpoint pathway that results in tumor immune evasion. In addition, ubiquitin-specific peptidase 7 (USP7) plays a crucial role in cancer development. In this study we investigated the role of USP7 in modulation of FGL1-mediated liver cancer immune evasion. We showed that knockdown of USP7 or treatment with USP7 inhibitor P5091 suppressed liver cancer growth by promoting CD8+ T cell activity in Hepa1-6 xenograft mice and in HepG2 or Huh7 cells co-cultured with T cells, whereas USP7 overexpression produced the opposite effect. We found that USP7 upregulated FGL1 in HepG2 and Huh7 cells by deubiquitination of transcriptional factor PR domain zinc finger protein 1 (PRDM1), which transcriptionally activated FGL1, and attenuated the CD8+ T cell activity, leading to the liver cancer growth. Interestingly, USP7 could be transcriptionally stimulated by PRDM1 as well in a positive feedback loop. P5091, an inhibitor of USP7, was able to downregulate FGL1 expression, thus enhancing CD8+ T cell activity. In an immunocompetent liver cancer mouse model, the dual blockade of USP7 and LAG3 resulted in a superior antitumor activity compared with anti-LAG3 therapy alone. We conclude that USP7 diminishes CD8+ T cell activity by a USP7/PRDM1 positive feedback loop on FGL1 production in liver cancer; USP7 might be a promising target for liver cancer immunotherapy.
Assuntos
Linfócitos T CD8-Positivos , Neoplasias Hepáticas , Peptidase 7 Específica de Ubiquitina , Regulação para Cima , Peptidase 7 Específica de Ubiquitina/metabolismo , Peptidase 7 Específica de Ubiquitina/antagonistas & inibidores , Peptidase 7 Específica de Ubiquitina/genética , Animais , Linfócitos T CD8-Positivos/imunologia , Linfócitos T CD8-Positivos/metabolismo , Humanos , Neoplasias Hepáticas/imunologia , Neoplasias Hepáticas/metabolismo , Neoplasias Hepáticas/patologia , Camundongos , Fator 1 de Ligação ao Domínio I Regulador Positivo/metabolismo , Fator 1 de Ligação ao Domínio I Regulador Positivo/genética , Linhagem Celular Tumoral , Camundongos Endogâmicos C57BL , Fibrinogênio , TiofenosRESUMO
In this retrospective study, we analyzed the expression of lymphocyte activating gene 3 (LAG-3) and fibrinogen-like protein 1 (FGP1) mRNA and the corresponding proteins in 78 patients with esophageal squamous cell carcinoma (ESCC) to evaluate the clinical significance and prognostic value. mRNA and protein expression were analyzed by reverse transcription PCR and Western blotting, respectively. The expression of LAG-3 and FGL1 mRNA and the corresponding proteins in tumor tissues were significantly increased in comparison with the normal esophageal mucosa. The overexpression of LAG-3 significantly correlated with the content of tumor-infiltrating lymphocytes (TILs), tumor differentiation, and TNM stage. The overexpression of FGL1 also significantly correlated with TILs, TNM stage, and lymph node metastasis. Kaplan-Meier survival analysis showed that tumor diameter, TNM stage, lymph node metastasis, LAG-3 and FGL1 protein expression were related to the progression-free survival (p<0.05). Multivariate Cox regression showed that the level of FGL1 and TNM stage were independent prognostic factors of progression-free survival. We speculated that the tumor microenvironment of ESCC induces immunosuppression due to up-regulated expression of LAG-3 and FGL1 in the tumor tissues, which promotes tumor growth.
Assuntos
Carcinoma de Células Escamosas , Neoplasias Esofágicas , Carcinoma de Células Escamosas do Esôfago , Humanos , Biomarcadores Tumorais/genética , Biomarcadores Tumorais/metabolismo , Carcinoma de Células Escamosas/diagnóstico , Carcinoma de Células Escamosas/genética , Carcinoma de Células Escamosas/metabolismo , Relevância Clínica , Neoplasias Esofágicas/diagnóstico , Neoplasias Esofágicas/genética , Carcinoma de Células Escamosas do Esôfago/genética , Carcinoma de Células Escamosas do Esôfago/patologia , Fibrinogênio/genética , Estimativa de Kaplan-Meier , Metástase Linfática , Linfócitos do Interstício Tumoral/patologia , Estadiamento de Neoplasias , Prognóstico , Estudos Retrospectivos , RNA Mensageiro/genética , RNA Mensageiro/metabolismo , Microambiente Tumoral/genéticaRESUMO
PURPOSE: To investigate the relationship between fibrinogen-like protein 1 (FGL-1) concentrations and various metabolic characteristics in patients with polycystic ovary syndrome (PCOS) and explore whether FGL-1 could be a predictive biomarker for PCOS. METHODS: This case-control study included 136 patients with PCOS and 34 normal controls recruited in the Department of Endocrinology and Metabolism, Shanghai Tenth People's Hospital between May 2017 and June 2021. Anthropometric characteristics, metabolic parameters, and reproductive hormones were collected. Serum FGL-1 measurement was conducted using enzyme-linked immunosorbent assay (ELISA) kits. RESULTS: Serum FGL-1 concentrations were higher in patients with PCOS than in control subjects in body mass index (BMI) subgroups, insulin resistance (IR) subgroups, and hepatic function subgroups, respectively. Serum FGL-1 concentrations were significantly associated with BMI, glycosylated hemoglobin A1c (HbA1c), fasting plasma glucose (FPG), homeostasis model assessment of insulin resistance (HOMA-IR), alanine aminotransferase (ALT), aspartate aminotransferase (AST), high-density lipoprotein cholesterol (HDL-c), and serum uric acid (SUA) in all individuals. The receiver operating characteristic (ROC) curve analysis revealed that the best cutoff value for FGL-1 levels to predict PCOS was 21.02 ng/ml with a sensitivity of 74.3% and a specificity of 70.6%. Both univariate and multiple logistic regressions indicated that the odds ratio (OR) for PCOS significantly increased in the subjects with high levels of FGL-1. CONCLUSION: In our study, FGL-1 was associated with serum aminotransferase and various metabolic indexes. Moreover, the high risk of PCOS was independently associated with the increased FGL-1 levels, which suggested that FGL-1 could be a predictive biomarker for PCOS.
Assuntos
Resistência à Insulina , Síndrome do Ovário Policístico , Alanina Transaminase , Aspartato Aminotransferases , Biomarcadores , Glicemia/análise , Índice de Massa Corporal , Estudos de Casos e Controles , China/epidemiologia , HDL-Colesterol , Feminino , Fibrinogênio , Hemoglobinas Glicadas/análise , Hormônios , Humanos , Insulina , Obesidade/complicações , Ácido ÚricoRESUMO
Glucosyl-galactosyl-hydroxylation (GGH) is one type of post-translational modification, which is mainly observed in collagen-like domain-containing proteins. Using LC-MS/MS analysis, we found a GGH-like modification at Lys65 of fibrinogen-like protein 1 (FGL1), although it does not contain a collagen-like domain. To identify the glycosyltransferases responsible for this modification, we established LH3/GLT25D1-knockout FGL1-overexpressing HT1080 cell lines. The result showed that knockout of LH3 or GLT25D1 significantly inhibited the glycosylation. Furthermore, deficiency of GGH by point mutation of the FGL1 protein or knockout of the GGH-related glycosyltransferase reduced FGL1 protein levels. Taken together, these data indicate that Lys65 of FGL1 is glucosyl-galactosyl-hydroxylated by LH3 and GLT25D1. Our results provide novel insights to regulate various FGL1 functions.
Assuntos
Fibrinogênio/metabolismo , Galactosiltransferases/metabolismo , Pró-Colágeno-Lisina 2-Oxoglutarato 5-Dioxigenase/metabolismo , Linhagem Celular Tumoral , Fibrinogênio/química , Glicosilação , Humanos , Lisina/metabolismo , Domínios Proteicos , Processamento de Proteína Pós-Traducional , Estabilidade ProteicaRESUMO
Immune checkpoint inhibition is an important strategy in cancer therapy. Blockade of CTLA-4 and PD-1/PD-L1 is well developed in clinical practice. In the last few years, LAG-3 has received much interest as an emerging novel target in immunotherapy. It was recently reported that FGL1 is a major ligand of LAG-3, which is normally secreted by the liver but is upregulated in several human cancers. FGL1 is a crucial biomarker and target for cancer immunotherapy. As the efficacy of immunotherapy is limited to specific types of patients, the subset of patients needs to be selected appropriately to receive precise treatment according to different biomarkers. To date, there is no test to accurately assess FGL1 expression levels. Nanobodies have some outstanding features, such as high stability, solubility and affinity for diagnostic and therapeutic applications. Here, we report the development and validation of a rapid, sensitive, and cost-effective nanobody-based immunoassay for the detection of FGL1 in human serum. In this study, human FGL1 recombinant protein was expressed and purified for the first time as an immunized antigen. Then, we constructed a nanobody phage display library and screened several nanobodies that bind FGL1 with high affinity. We selected two nanobodies targeting different epitopes of FGL1, one as a capture and the other conjugated with HRP as a probe. The double nanobody-based sandwich ELISA to detect the concentration of FGL1 showed a good response relationship in the range of 15.625-2000 ng/mL, and the recoveries from the spiked sample were in the range of 78% and 100%. This assay could be used as a potential approach for evaluating FGL1 expression for patient stratification and for predicting the therapeutic efficacy of targeting the LAG3/FGL1 axis.
Assuntos
Fibrinogênio/imunologia , Fibrinogênio/metabolismo , Anticorpos de Domínio Único/imunologia , Anticorpos de Domínio Único/metabolismo , Animais , Camelus , Ensaio de Imunoadsorção Enzimática/métodos , Células HEK293 , Humanos , Imunoensaio/métodosRESUMO
The combination of an immuno-metabolic adjuvant and immune checkpoint inhibitors holds great promise for effective suppression of tumor growth and invasion. In this study, a pH-responsive co-delivery platform was developed for metformin (Met), a known immuno-metabolic modulator, and short interfering RNA (siRNA) targeting fibrinogen-like protein 1 mRNA (siFGL1), using a hybrid biomimetic membrane (from macrophages and cancer cells)-camouflaged poly (lactic-co-glycolic acid) nanoparticles. To improve the endo-lysosomal escape of siRNA for effective cytosolic siRNA delivery, a pH-triggered CO2 gas-generating nanoplatform was developed using the guanidine group of Met. It can react reversibly with CO2 to form Met-CO2 for the pH-dependent capture/release of CO2. The introduction of Met, a conventional anti-diabetic drug, promotes programmed death-ligand 1 (PD-L1) degradation by activating adenosine monophosphate-activated protein kinase, subsequently blocking the inhibitory signals of PD-L1. As a result, siFGL1 delivery by the camouflaged nanoparticles of the hybrid biomimetic membrane can effectively silence the FGL1 gene, promoting T-cell-mediated immune responses and enhancing antitumor immunity. We found that a combination of PD-L1/programmed death 1 signaling blockade and FGL1 gene silencing exhibited high synergistic therapeutic efficacy against breast cancer in vitro and in vivo. Additionally, Met alleviated tumor hypoxia by reducing oxygen consumption and inducing M1-type differentiation of tumor-related macrophages, which improved the tumor immunosuppressive microenvironment. Our results indicate the potential of hybrid biomimetic membrane-camouflaged nanoparticles and combined Met-FGL1 blockade in breast cancer immunotherapy.
Assuntos
Antineoplásicos/farmacologia , Neoplasias da Mama/tratamento farmacológico , Imunossupressores/farmacologia , Imunoterapia/métodos , Nanopartículas/química , Microambiente Tumoral/efeitos dos fármacos , Animais , Apoptose , Antígeno B7-H1 , Biomimética , Mama , Neoplasias da Mama/patologia , Ciclo Celular , Linhagem Celular Tumoral , Citocinas , Feminino , Fibrinogênio/genética , Inativação Gênica/efeitos dos fármacos , Humanos , Concentração de Íons de Hidrogênio , Lisossomos , Macrófagos/metabolismo , Metformina , Camundongos , Camundongos Endogâmicos BALB C , Células RAW 264.7 , RNA Interferente Pequeno , Transdução de Sinais/efeitos dos fármacosRESUMO
BACKGROUND: Fibrinogen-like protein 1 (FGL1)-Lymphocyte activating gene 3 (LAG-3) pathway is a promising immunotherapeutic target and has synergistic effect with programmed death 1 (PD-1)/programmed death ligand 1 (PD-L1). However, the prognostic significance of FGL1-LAG-3 pathway and the correlation with PD-L1 in hepatocellular carcinoma (HCC) remain unknown. METHODS: The levels of LAG-3, FGL1, PD-L1 and cytotoxic T (CD8+T) cells in 143 HCC patients were assessed by multiplex immunofluorescence. Associations between the marker's expression and clinical significances were studied. RESULTS: We found FGL1 and LAG-3 densities were elevated while PD-L1 and CD8 were decreased in HCC tissues compared to adjacent normal liver tissues. High levels of FGL1 were strongly associated with high densities of LAG-3+cells but not PD-L1. CD8+ T cells densities had positive correlation with PD-L1 levels and negative association with FGL1 expression. Elevated densities of LAG-3+cells and low levels of CD8+ T cells were correlated with poor disease outcome. Moreover, LAG-3+cells deteriorated patient stratification based on the abundance of CD8+ T cells. Patients with positive PD-L1 expression on tumor cells (PD-L1 TC+) tended to have an improved survival than that with negative PD-L1 expression on tumor cells (PD-L1 TC-). Furthermore, PD-L1 TC- in combination with high densities of LAG-3+cells showed the worst prognosis, and PD-L1 TC+ patients with low densities of LAG-3+cells had the best prognosis. CONCLUSIONS: LAG-3, FGL1, PD-L1 and CD8 have distinct tissue distribution and relationships with each other. High levels of LAG-3+cells and CD8+ T cells represent unfavorable and favorable prognostic biomarkers for HCC respectively.
Assuntos
Carcinoma Hepatocelular , Neoplasias Hepáticas , Antígeno B7-H1 , Linfócitos T CD8-Positivos , Fibrinogênio , Humanos , PrognósticoRESUMO
Non-small cell lung cancer (NSCLC) is characterized by a high incidence rate and poor prognosis worldwide. A deeper insight into the pathogenesis of NSCLC and identification of novel therapeutic targets are essential to improve the prognosis of NSCLC. In this study, we revealed that fibrinogen-like protein 1 (FGL1) promotes proliferation, migration, and invasion of NSCLC cells. Mechanistically, we found that Stat3 acts as a transcription factor and can be recruited to the FGL1 promoter, enhancing FGL1 promoter activity. Lysine-specific demethylase 4A (KDM4A) interacts with Stat3 and facilitates the removal of methyl groups from H3K9me3, thereby enhancing Stat3-mediated transcription of FGL1. Furthermore, we observed that Stat3 and KDM4A promote NSCLC cell proliferation, migration, and invasion partly by upregulating FGL1 expression. Additionally, the expression of FGL1 was significantly higher in cancer tissues (n = 90) than in adjacent non-cancerous tissues (n = 90). Furthermore, patients with high FGL1 expression had a shorter overall survival (OS) compared to those with low FGL1 expression. We measured the expression levels of FGL1 on circulating tumor cells (CTCs) in 65 patients and found that patients with a dynamic decrease in FGL1 expression on CTCs exhibited a better therapeutic response. These findings suggest that the dynamic changes in FGL1 expression can serve as a potential biomarker for predicting treatment efficacy in NSCLC. Overall, this study revealed the significant role and regulatory mechanisms of FGL1 in the development of NSCLC, suggesting its potential as a therapeutic target for patients with NSCLC. Future studies should provide more personalized and effective treatment options for patients with NSCLC to improve clinical outcomes.
Assuntos
Biomarcadores Tumorais , Carcinoma Pulmonar de Células não Pequenas , Progressão da Doença , Histona Desmetilases com o Domínio Jumonji , Neoplasias Pulmonares , Fator de Transcrição STAT3 , Humanos , Carcinoma Pulmonar de Células não Pequenas/genética , Carcinoma Pulmonar de Células não Pequenas/patologia , Carcinoma Pulmonar de Células não Pequenas/metabolismo , Neoplasias Pulmonares/genética , Neoplasias Pulmonares/patologia , Neoplasias Pulmonares/metabolismo , Fator de Transcrição STAT3/metabolismo , Fator de Transcrição STAT3/genética , Biomarcadores Tumorais/metabolismo , Biomarcadores Tumorais/genética , Camundongos , Animais , Histona Desmetilases com o Domínio Jumonji/metabolismo , Histona Desmetilases com o Domínio Jumonji/genética , Feminino , Metástase Neoplásica , Fibrinogênio/metabolismo , Fibrinogênio/genética , Masculino , Linhagem Celular Tumoral , Proliferação de Células , Movimento Celular , Prognóstico , Regulação Neoplásica da Expressão GênicaRESUMO
Liver disease is a complex group of diseases with high morbidity and mortality rates, emerging as a major global health concern. Recent studies have highlighted the involvement of fibrinogen-like proteins, specifically fibrinogen-like protein 1 (FGL1) and fibrinogen-like protein 2 (FGL2), in the regulation of various liver diseases. FGL1 plays a crucial role in promoting hepatocyte growth, regulating lipid metabolism, and influencing the tumor microenvironment (TME), contributing significantly to liver repair, non-alcoholic fatty liver disease (NAFLD), and liver cancer. On the other hand, FGL2 is a multifunctional protein known for its role in modulating prothrombin activity and inducing immune tolerance, impacting viral hepatitis, liver fibrosis, hepatocellular carcinoma (HCC), and liver transplantation. Understanding the functions and mechanisms of fibrinogen-like proteins is essential for the development of effective therapeutic approaches for liver diseases. Additionally, FGL1 has demonstrated potential as a disease biomarker in radiation and drug-induced liver injury as well as HCC, while FGL2 shows promise as a biomarker in viral hepatitis and liver transplantation. The expression levels of these molecules offer exciting prospects for disease assessment. This review provides an overview of the structure and roles of FGL1 and FGL2 in different liver conditions, emphasizing the intricate molecular regulatory processes and advancements in targeted therapies. Furthermore, it explores the potential benefits and challenges of targeting FGL1 and FGL2 for liver disease treatment and the prospects of fibrinogen-like proteins as biomarkers for liver disease, offering insights for future research in this field.
RESUMO
LAG3 is an inhibitory immune checkpoint expressed on activated T and NK cells. Blocking the interaction of LAG3 with its ligands MHC-II and FGL1 renders T cells improved cytotoxicity to cancer cells. Current study generated a panel of LAG3 monoclonal antibodies (mAbs) through immunization of mice followed by phage display. Some of them bound to the D1-D2 domain of LAG3, which is known for the engagement of its ligands FGL1 and MHC-II. Three outperformers, M208, M226, and M234, showed stronger blocking activity than Relatlimab in the FGL1 binding. Furthermore, M234 showed dual inhibition of FGL1 (IC50 of 20.6â¯nM) and MHC-II binding (IC50 of 6.2â¯nM) to LAG3. In vitro functional tests showed that M234 significantly stimulated IFN-γ secretion from activated PBMC cells. In vivo studies in a mouse model of hepatocellular carcinoma xenografts demonstrated that combining M234 IgG with GPC3-targeted bispecific antibodies significantly improved efficacy. In addition, GPC3-targeted CAR-T cells secreting IL-21-M234 scFv fusion protein exhibited enhanced activity in inhibiting tumor growth and greatly increased the survival rate of mice. Taken together, M234 has potential in cancer immunotherapy and warrants further clinical trial.
Assuntos
Anticorpos Neutralizantes , Antígenos CD , Imunoterapia , Proteína do Gene 3 de Ativação de Linfócitos , Animais , Humanos , Camundongos , Antígenos CD/imunologia , Antígenos CD/metabolismo , Anticorpos Neutralizantes/farmacologia , Anticorpos Neutralizantes/imunologia , Ligantes , Imunoterapia/métodos , Linhagem Celular Tumoral , Antígenos de Histocompatibilidade Classe II/imunologia , Antígenos de Histocompatibilidade Classe II/metabolismo , Ensaios Antitumorais Modelo de Xenoenxerto , Neoplasias Hepáticas/imunologia , Neoplasias Hepáticas/tratamento farmacológico , Neoplasias Hepáticas/patologia , Neoplasias Hepáticas/terapia , Carcinoma Hepatocelular/imunologia , Carcinoma Hepatocelular/tratamento farmacológico , Carcinoma Hepatocelular/terapia , Carcinoma Hepatocelular/patologia , Camundongos Endogâmicos BALB C , Ligação Proteica , Feminino , Anticorpos Monoclonais/farmacologiaRESUMO
Aside from antibodies, peptides show great potential as immune checkpoint inhibitors (ICIs) due to several advantages, such as better tumor penetration and lower cost. Lymphocyte-activation gene 3 (LAG-3) is an immune checkpoint which can induce T cell dysfunction through interaction with its soluble ligand fibrinogen like protein-1 (FGL1). Here, we found that LAG-3 expression was higher than programmed cell death protein 1 (PD-1) in multiple human cancers by TCGA databases, and successfully identified a LAG-3 binding peptide LFP-6 by phage display bio-panning, which specifically blocks the interaction of LAG-3/FGL1 but not LAG-3/MHC-II. Subsequently, d-amino acids were introduced to substitute the N- and C-terminus of LFP-6 to obtain the proteolysis-resistant peptide LFP-D1, which restores T cell function in vitro and inhibits tumor growth in vivo. Further, a bispecific peptide LFOP targeting both PD-1/PD-L1 and LAG-3/FGL1 was designed by conjugating LFP-D1 with PD-1/PD-L1 blocking peptide OPBP-1(8-12), which activates T cell with enhanced proliferation and IFN-γ production. More importantly, LFOP combined with radiotherapy significantly improve the T cell infiltration in tumor and elevate systemic antitumor immune response. In conclusion, we developed a novel peptide blocking LAG-3/FGL1 which can restore T cell function, and the bispecific peptide synergizes with radiotherapy to further enhance the antitumor immune response.
RESUMO
Partial hepatectomy is a first-line treatment for hepatocellular carcinoma. Within 2 weeks following partial hepatectomy, specific molecular pathways are activated to promote liver regeneration. Nevertheless, residual microtumors may also exploit these pathways to reappear and metastasize. Therapeutically targeting molecules that are differentially regulated between normal cells and malignancies, such as fibrinogen-like protein 1 (FGL1), appears to be an effective approach. The potential functions of FGL1 in both regenerative and malignant cells are discussed within the ambit of this review. While FGL1 is normally elevated in regenerative hepatocytes, it is normally downregulated in malignant cells. Hepatectomy does indeed upregulate FGL1 by increasing the release of transcription factors that promote FGL1, including HNF-1α and STAT3, and inflammatory effectors, such as TGF-ß and IL6. This, in turn, stimulates certain proliferative pathways, including EGFR/Src/ERK. Hepatectomy alters the phase transition of highly differentiated hepatocytes from G0 to G1, thereby transforming susceptible cells into cancerous ones. Activation of the PI3K/Akt/mTOR pathway by FGL1 allele loss on chromosome 8, a tumor suppressor area, may also cause hepatocellular carcinoma. Interestingly, FGL1 is specifically expressed in the liver via HNF-1α histone acetylase activity, which triggers lipid metabolic reprogramming in malignancies. FGL1 might also be involved in other carcinogenesis processes such as hypoxia, epithelial-mesenchymal transition, immunosuppression, and sorafenib-mediated drug resistance. This study highlights a research gap in these disciplines and the necessity for additional research on FGL1 function in the described processes.
RESUMO
Immune escape is the major reason for immunotherapy failure in stomach adenocarcinoma (STAD). We tried to reveal the underlying mechanism of FGL1 influencing STAD in this study. Bioinformatics analyses were conducted to analyze the expression of FGL1, the signaling pathways affected by FGL1, and the relation between FGL1 and immune cell infiltration. Quantitative real-time PCR (qRT-PCR), cell counting kit-8 assay, colony formation assay, flow cytometry and Transwell assay were adopted to analyze FGL1 expression, cell viability, cell proliferation, cell apoptosis, and cell invasion, respectively. Enzyme-linked immunosorbent assay, lactate dehydrogenase method, qRT-PCR and Western blot were adopted to reveal proinflammatory cytokine expression, cytotoxicity and mRNA and protein expression of the Notch signaling-related genes, respectively, after co-culture of STAD cells and CD8+T cells. Nude mice experiment was conducted to validate the results obtained above. FGL1 expressed highly in STAD and could activate the Notch signaling pathway, and it was negatively correlated with CD8+T cell infiltration. Cell experiments confirmed that high expression of FGL1 facilitated proliferation and hindered apoptosis of STAD cells. Knockdown of FGL1 could facilitate expression of pro-inflammatory factors and the cytotoxicity of CD8+T cells in co-culture system of STAD and CD8+ T cells. Knockdown of FGL1 could suppress the expression of the Notch signaling pathway-related genes, and the addition of Notch inhibitor proved that FGL1 promoted immune escape via the Notch signaling pathway. This study investigated the influence of FGL1 on STAD immune escape and demonstrated that FGL1 inhibited CD8+ T cell activation by activating the Notch signaling pathway and thus promoted tumor immune escape in STAD, providing a new potential diagnostic marker and therapeutic target for the immunotherapy of STAD patients.
RESUMO
Upregulation of FGL1 helps tumors escape from immune surveillance, and therapeutic antibodies targeting FGL1 have potential as another immune checkpoint inhibitor. However, the underlying mechanism of high FGL1 protein level in cancers is not well defined. Here, we report that FBXO38 interacts with and ubiquitylates FGL1 to negatively regulate its stability and to mediate cancer immune response. Depletion of FBXO38 markedly augments FGL1 abundance, not only suppressing CD8+ T cell infiltration and enhancing immune evasion of tumor but also increasing inflammation in mice. Importantly, we observe a negative correlation of FBXO38 with FGL1 and IL-6 in non-small cell lung cancer specimens. FGL1 and IL-6 levels positively correlate with TNM (tumor, lymph node, metastasis) stages, while FBXO38 and the infiltrating CD8+ T cells negatively correlate with TNM stages. Our study identifies a mechanism regulating FGL1 stability and a target to enhance the immunotherapy and suggests that the combination of anti-FGL1 and anti-IL-6 is a potential therapeutic strategy for cancer immunotherapy.