Abstract Background and aims The immunosuppressive tumor microenvironment (TME) plays an essential role in cancer progression and immunotherapy response. Despite the considerable advancements in cancer immunotherapy, the limited response to immune checkpoint blockade (ICB) therapies in patients with hepatocellular carcinoma (HCC) remains a major challenge for its clinical implications. Here, we investigated the molecular basis of the protein O-fucosyltransferase 1 (POFUT1) that drives HCC immune evasion and explored a potential therapeutic strategy for enhancing ICB efficacy. Methods De novo MYC/Trp53^−/− liver tumor and the xenograft tumor models were used to evaluate the function of POFUT1 in immune evasion. Biochemical assays were performed to elucidate the underlying mechanism of POFUT1-mediated immune evasion. Results We identified POFUT1 as a crucial promoter of immune evasion in liver cancer. Notably, POFUT1 promoted HCC progression and inhibited T-cell infiltration in the xenograft tumor and de novo MYC/Trp53^−/− mouse liver tumor models. Mechanistically, we demonstrated that POFUT1 stabilized programmed death ligand 1 (PD-L1) protein by preventing tripartite motif containing 21-mediated PD-L1 ubiquitination and degradation independently of its protein-O-fucosyltransferase activity. In addition, we further demonstrated that PD-L1 was required for the tumor-promoting and immune evasion effects of POFUT1 in HCC. Importantly, inhibition of POFUT1 could synergize with anti-programmed death receptor 1 therapy by remodeling TME in the xenograft tumor mouse model. Clinically, POFUT1 high expression displayed a lower response rate and worse clinical outcome to ICB therapies. Conclusions Our findings demonstrate that POFUT1 functions as a novel regulator of tumor immune evasion and inhibition of POFUT1 may be a potential therapeutic strategy to enhance the efficacy of immune therapy in HCC. Keywords: Hepatocellular Carcinoma, Immune Checkpoint Inhibitor, Tumor microenvironment - TME __________________________________________________________________ WHAT IS ALREADY KNOWN ON THIS TOPIC * Hepatocellular carcinoma (HCC) is the main primary liver cancer with a poor prognosis. Immune checkpoint blockade (ICB) therapies, such as programmed death receptor 1 (PD-1) antibody therapy and programmed death ligand 1 (PD-L1) antibody therapy, have been approved therapy for patients with HCC, but limited survival benefits have been observed due to tumor immune evasion. Protein O-fucosyltransferase 1 (POFUT1) expression is elevated in several human cancers, but its role in HCC progression and immunosuppressive microenvironment is unclear. WHAT THIS STUDY ADDS * This study uncovers a new functional link between POFUT1 and immune evasion in HCC. POFUT1 stabilizes PD-L1 protein by preventing tripartite motif containing 21-mediated ubiquitination and degradation, leading to an immunosuppressive tumor microenvironment to favor HCC progression. Inhibition of POFUT1 can synergize with anti-PD-1 therapy in the HCC mouse model. HOW THIS STUDY MIGHT AFFECT RESEARCH, PRACTICE OR POLICY * This study elucidates the crucial role of POFUT1 in liver cancer immune evasion and provides an efficient strategy to potentiate “cold” tumor’s response to immune checkpoint blockade therapies. Introduction Primary liver cancer is the fourth leading cause of cancer death worldwide.[43]^1 Hepatocellular carcinoma (HCC) accounts for the majority of primary liver cancers and has a very poor prognosis.[44]^2 3 Recently, immune checkpoint blockade (ICB) therapies, such as programmed death receptor 1 (PD-1) antibody therapy and programmed death ligand 1 (PD-L1) antibody therapy, have been approved therapy for patients with HCC.[45]4,[46]6 However, only limited survival benefits for patients with HCC have been observed owing to tumor immune evasion.[47]^7 8 Thus, it is meaningful to gain insight into the mechanism of tumor immune evasion to improve ICB therapy. PD-L1 is often overexpressed on the surface of cancer cells.[48]^9 The binding of PD-L1 and PD-1 inhibits T-cell proliferation and activation, suppresses the function of cytotoxic T cells, and ultimately induces immune escape and tumor development, leading to immunotherapy failure.[49]^10 11 Clinically, PD-L1 expression level in tumors is an important biomarker to evaluate the efficacy of patients for ICB therapies, but currently, studies report that both PD-L1-positive and PD-L1-negative patients show promising responses to immunotherapy.[50]12,[51]14 Emerging evidence has shown that downregulating the PD-L1 expression in tumors can improve the efficacy of anti-PD-1 therapy.[52]15,[53]18 Increased PD-L1 expression is frequently used by tumor cells as a strategy to evade antitumor immune responses.[54]^19 Therefore, exploring the mechanism of aberrant PD-L1 expression in tumor is crucial to resolve the conflicting observations and enhance clinical responses to ICB therapy. Emerging evidence has indicated that glycosylation of immune receptors and ligands plays a critical role in cancer immunity.[55]^20 Protein glycosylation consists mainly of N-glycosylation and O-glycosylation.[56]^21 Currently, the role of N-glycosylation-related genes in tumor immune escape has drawn increasing attention[57]22,[58]24; however, the functions of O-glycosylation-related genes in cancer progression remain unclear. Fucosylation is a type of glycosylation modification and fucosylated glycan structures are commonly present on the cell surface. There are 13 fucosyltransferase genes in the human genome. Protein O-fucosyltransferases (POFUTs) are located in the endoplasmic reticulum and catalyze O-linked fucosylation.[59]^25 Emerging evidence has suggested that the expression of POFUT1 is elevated in several human cancers,[60]^26 27 however, whether and how POFUT1 plays an essential role in HCC progression and immunosuppressive microenvironment remains unclear. In this study, we have identified a previously unknown functional link between POFUT1 and immune evasion in HCC. POFUT1 stabilizes PD-L1 protein by preventing the tripartite motif containing 21 (TRIM21)-mediated degradation and ubiquitination, leading to HCC progression and immune microenvironment suppression. From a therapeutic viewpoint, our findings revealed that inhibition of POFUT1 synergized with anti-PD-1 therapy in the HCC mouse model. Thus, this study provides a potential therapeutic approach for HCC immunotherapy. Materials and methods Plasmids and reagents PT3-EF1a-MYC-IRES-luciferase (MYC), px330-sg-p53 (sg-p53) and CMV-SB13 were kindly provided by Amaia Lujambio, Icahn School of Medicine at Mount Sinai. DNA fragments of POFUT1 and PD-L1 were cloned into MYC by replacing the luciferase sequence to generate MYC-POFUT1 and MYC-PD-L1. Additional information related to plasmids and reagents can be found in the [61]online supplemental information. Cell dissociation and flow cytometry analysis Mouse tumor tissues were harvested and immediately placed into a centrifuge tube on ice. The tumor tissue was then washed with phosphate-buffered saline and cut into small pieces using scissors. Collagenase I was used for enzymatic digestion with constant shaking at 37°C for 20–30 min. The enzymatic reaction was subsequently terminated, and the sample was filtered through a 70 micron cell strainer to remove undissociated tissue fragments. For flow cytometry, approximately 1×10ˆ5 to 1×10ˆ6 cells per sample were stained and subsequently analyzed with a BD Fortessa flow cytometer (BD Biosciences). Clinical samples and immunohistochemistry The patient samples used in this study were 90 liver cancer tissue specimens collected between 2014 and 2018 from Shanghai Renji Hospital, affiliated with Shanghai Jiao Tong University School of Medicine, including 72 men and 18 women with an age range of 33–83 years. Tissue microarrays were prepared by Shanghai Zuocheng Bio company. Informed consent was obtained from each patient before sample collection. All research was conducted according to the principles outlined in the Helsinki declaration and the Istanbul declaration, and the use of these samples and informed consent were approved by the Ethics Committee of Renji Hospital, Shanghai Jiao Tong University School of Medicine (RA-2020–250). One case was excluded from statistical analysis due to sample detachment during tissue sectioning. Immunohistochemical (IHC) staining was conducted in accordance with established protocols. Specifically, the process included deparaffinization and rehydration, followed by antigen retrieval using citrate buffer on paraffin-embedded sections. Subsequently, the sections were blocked and incubated with the corresponding primary antibody. Detection was achieved through the application of the 3,3’-diaminobenzidine substrate. For estimating the number of positive cells: a box with an area of 0.04 mm^2 was drawn using Aperio software, and the number of positive cells was counted at 20× magnification. The number was then multiplied by 25 to estimate the number per mm^2. For tissue microarrays, the extent of staining was quantified using a “Staining Area (%)” score (0=no positive staining; 1=1–25%; 2=26–50%; 3=51–75%; 4=76–100%), and staining intensity was evaluated with an “Intensity Score” (0=no staining; 1=weak; 2=mild; 3=moderate; 4=strong; 5=intense). Animal experiments Hydrodynamic tail-vein injection For the experiments to detect the functionality of POFUT1, we prepared a 2 mL solution of 0.9% sodium chloride containing 12 µg of pT3-EF1a-MYC-IRES-luciferase (MYC), 10 µg of px330-sg-p53 (sg-p53), 12 µg of a mixture of two lenti-CRISPR sgPofut1 (or 12 µg of control plasmid lenti-CRISPR V2), and 6 µg of the transposon SB13 transposase-encoding plasmid. For the experiments to detect the overexpression functionality of POFUT1, we prepared a 2 mL solution of 0.9% sodium chloride containing 12 µg of pT3-EF1a-MYC-POFUT1 (or 12 µg of MYC), 10 µg of sg-p53, and 6 µg of the transposon SB13 transposase-encoding plasmid. For the experiments to detect the functionality of PD-L1, the first group was prepared with a 2 mL solution of 0.9% sodium chloride containing 12 µg of pT3-EF1a-MYC-PD-L1 (or 12 µg of control plasmid MYC), 10 µg of sg-p53, 12 µg of a mixture of two lenti-CRISPR sgPofut1 (or 12 µg of control plasmid lenti-CRISPR V2), and 6 µg of the transposon SB13 transposase-encoding plasmid. The second group was prepared with a 2 mL solution of 0.9% sodium chloride containing 12 µg of pT3-EF1a-MYC-POFUT1 (or 12 µg of control plasmid MYC), 10 µg of sg-p53, 12 µg of a mixture of two lenti-CRISPR sgPdl1 (or 12 µg of control plasmid lenti-CRISPR V2), and 6 µg of the transposon SB13 transposase-encoding plasmid. The mice were intravenously injected with the 0.9% sodium chloride/plasmid mixture through the tail vein. The injection volume was 10% of their body weight, and the injection was completed within 5–7 s. Tumor xenograft mouse models Subcutaneous injections of 1×10ˆ6 Hepa1-6 cells were administered into the inguinal region of the mice. The order of subcutaneous tumor measurement is randomized. After approximately 2 weeks, mice were euthanized using cervical dislocation, and the tumors were excised for subsequent analysis. Tumor volume was calculated using the formula length×width^2/2. For the PD-1 treatment experiment, intraperitoneal injection of anti-PD-1 antibody (100 µg per mouse, Bio X Cell, BE0146) or IgG isotype control was performed every 3 days for a total of four injections. For depletion of CD8^+ T cells in vivo, mice were intraperitoneally injected with anti-CD8α antibodies (200 µg per mouse, Bio X Cell, BE0061) or IgG isotype control 2 days before tumor implantation and two times per week thereafter for a total of five injections to ensure sustained depletion of CD8^+ T cells during the experimental period. Male C57 mice (5–6 weeks old, obtained from [62]www.jh-labanimal.site/) were used in this study. Experimental animals that fail to receive successful hydrodynamic tail vein injection or subcutaneous tumor implantation, or die prematurely before the designated time after successful modeling (injecting the hydrodynamic tail vein too quickly can lead to the death of the mouse, occurring within 6 hours after injection, unrelated to tumor occurrence) are not included in the experimental statistics. Each group of experimental animals has similar body weights, and the animals are randomly assigned to groups using a computer-based random sequence generator. Measurements are randomized in order. During tumor harvesting, one researcher erases grouping information, and another researcher who is unaware of the grouping is responsible for collecting and processing the samples as well as subsequent data analysis. All animal experiments were conducted in accordance with relevant guidelines and regulations and received approval from the Animal Experimental Committee of Renji Hospital, Shanghai Jiao Tong University School of Medicine (RJ20220720). Statistical analysis The statistical analysis was performed using GraphPad Prism V.9 software. Flow cytometry data were analyzed using FlowJo_V.10 software. The biological informatics analysis was conducted using the R language (V.4.1.1). Gene Set Enrichment Analysis (GSEA) was conducted using either R language or GSEA software. Quantification of western blot and IHC was performed using ImageJ software. Data availability RNA sequencing (RNA-seq) data are accessible at the NODE under accession number: OEP004799 ([63]https://www.biosino.org/node/project/detail/OEP004799). [64]GSE22058, [65]GSE14520 and ICGC_JP data sets were downloaded from the HCCDB website ([66]http://lifeome.net/database/hccdb/home.html).[67]^28 The Cancer Genome Atlas Liver hepatocellular carcinoma (TCGA_LIHC) data set was obtained through the UCSC website ([68]https://xenabrowser.net/datapages/). The patients with Chinese HCC with hepatitis B virus (HBV) infection (CHCC_HBV) cohort was downloaded from the CPTAC website ([69]https://proteomics.cancer.gov/programs/cptac).[70]^29 Tumor immune scores and microenvironment scores were evaluated and downloaded from the TIMER V.2.0 website ([71]http://timer.cistrom.org/).[72]^30 The immune subtypes were obtained from Bagaev et al.[73]^31 The leukocyte and stromal fractions and the scores of lymphocyte infiltration, Th1 cells, Th2 cells and proliferation were obtained from Thorsson et al.[74]^32 The gene sets of expanded interferon (IFN)-G signature, a good response associated genes 1 and up in responders to anti-cytotoxic T-lymphocyte associated protein-4+anti-PD-1 were obtained from Cerezo-Walli et al.[75]^33 The genes related to IFN-G signaling, effector T-cell signaling, chemokine and antigen presentation pathway were obtained from Newell et al.[76]^34 All data used in this study are publicly available for download. All data are available on request from the authors. Additional detailed Methods are included in [77]online supplemental information. Results POFUT1 is upregulated in HCC and correlates with immunosuppressive microenvironment To identify the O-glycosylation-related genes that play an important role in HCC immunosuppressive microenvironment, we first explored the genes both upregulated in TCGA_LIHC and CHCC_HBV_2019 (CHCC_HBV) cohorts (logFC>0.5, p value<0.05) and found that 29 of them were O-glycosylation-related genes ([78]figure 1A). Second, we estimated the relationship between the 29 candidate genes and tumor immune microenvironment by xCell method in TCGA_LIHC and found the top enrichment of POFUT1 in the poor immune score ([79]figure 1B). Similar results were obtained in the CHCC_HBV and three other HCC databases (HCCB1_GSE22058, HCCB6_GSE14520 and HCCB18_ICGC_JP) ([80]figure 1C,D and [81]online supplemental figure S1A). Moreover, we further examined the microenvironment score for the identified candidate genes and also found high enrichment of POFUT1 in the context of a poor microenvironment score ([82]figure 1E and [83]online supplemental figure S1B). Due to its substantial enrichment, POFUT1 was selected for further investigation into its functional link with HCC immunosuppressive microenvironment. We further investigated whether POFUT1 could regulate the infiltration and response of immune cells. We first performed the correlation analysis between POFUT1 expression and scores of immune cells based on xCell algorithm, and found that POFUT1 expression was negatively correlated with the infiltration of many antitumor-related immune cells in TCGA_LIHC ([84]figure 1F). Additionally, immune-related analyses were performed on the POFUT1 high and low expression groups within the TCGA_LIHC cohort. The result indicated a decrease in the leukocyte fraction, and the scores of lymphocyte infiltration and Th1 cells were also diminished. In contrast, the scores of Th2 and proliferation were increased in the POFUT1 high-expression group ([85]figure 1G). Moreover, we analyzed the tumor immune subtype proportion between POFUT1 high and low expression groups in the TCGA_LIHC cohort and found that POFUT1 high expression group tended to exhibit more D (immune-depleted) and F (fibrotic) phenotypes, conversely, more IE (immune-enriched/non-fibrotic) and IE/IF (immune-enriched/fibrotic) phenotype were found in the POFUT1 low expression group ([86]figure 1H). GSEA also showed that immune-related pathways were enriched in POFUT1 low expression group ([87]online supplemental figure S1C). We next analyzed the correlation between POFUT1 expression and the IFN-G signaling, effector T-cell signaling, chemokine and antigen presentation pathway in TCGA_LIHC and CHCC_HBV cohorts, and the universal negative relationship was found in the POFUT1 and above factors ([88]figure 1I). Additionally, GSEA revealed that the expanded IFN-G signature was enriched in the POFUT1 low expression group ([89]online supplemental figure S1D). Overall, these results suggest that POFUT1 high expression may contribute to the immunosuppressive microenvironment in HCC and the immune escape of tumor cells. Figure 1. POFUT1 is highly expressed in HCC and is associated with the immunosuppressive microenvironment. (A) The Venn diagram showed that there were 29 genes upregulated in both TCGA_LIHC (n=369) and CHCC_HBV (n=159) (tumor vs normal, p value<0.05, log fold change >0.5) that intersected with the gene set GOBP_PROTEIN_O_LINKED_GLYCOSYLATION. (B–C) Volcano plot showing the distribution of immune score differences for 29 candidate genes grouped by expression level (high vs low) in TCGA_LIHC (n=369) and CHCC_HBV (n=159). (D–E) Volcano plot showing the distribution of immune score and microenvironment score differences for 29 candidate genes grouped by expression level (high vs low) in HCCB18_ICGC_JP (n=212). (F) The correlation heatmap for POFUT1 expression and scores of different types of immune cells in the HCC data sets (based on xCell algorithm). The Spearman coefficient was used to evaluate correlations. (G) Leukocyte fraction and stromal fraction, scores of lymphocyte infiltration, scores of Th1 and Th2 cells and score of proliferation with high or low POFUT1 in TCGA_LIHC (n=369). Data represent mean±SD. P values were calculated using Student’s t-test. (H) Distribution of immune subtypes with high or low POFUT1 in TCGA_LIHC (n=369). (I) Heatmap of correlation between POFUT1 expression and IFN-G signaling pathway, effector T-cell signaling pathway, chemokine pathway and MHC-I pathway in TCGA_LIHC (n=369) and CHCC_HBV (n=159). The Pearson coefficient was used to evaluate correlations. *p<0.05; **p<0.01; ***p<0.001; ****p<0.0001. CHCC, Chinese HCC; D, immune depleted; F, fibrotic; HBV, hepatitis B virus; HCC, hepatocellular carcinoma; IE, immune-enrichment/non-fibrotic; IE/IF, immune-enrichment/fibrotic; IFN, interferon; MHC, major histocompatibility complex; POFUT1, protein O-fucosyltransferase 1; TCGA_LIHC, The Cancer Genome Atlas Liver Hepatocellular Carcinoma. [90]Figure 1 [91]Open in a new tab Downregulation of POFUT1 inhibits HCC progression in vivo and improves tumor immunosuppressive microenvironment To investigate the role of POFUT1 in HCC progression, we first constructed the Pofut1 knockdown cell line in mouse hepatoma cell Hepa1-6. The levels of Pofut1 protein and messenger RNA (mRNA) were determined by western blot and quantitative PCR, respectively ([92]online supplemental figure S2A,B). Then, we injected the Hepa1-6 cells subcutaneously into C57BL/6 mice to confirm the function of Pofut1 in vivo. Genetic knockdown of Pofut1 expression significantly inhibited liver tumor growth in C57BL/6 mice ([93]figure 2A,B). Next, we generated a mouse MYC/Trp53^−/− HCC model[94]^35 36 following hydrodynamic tail vein injections of MYC, sg-p53 and CMV-SB13 vectors. We delivered a Pofut1 targeting sgRNA expressing vector (or control vector) in combination with MYC, sg-p53 and CMV-SB13 ([95]figure 2C). sgPofut1 mice exhibited less aggressive HCC development 35 days after hydrodynamic injection, showing fewer tumors and smaller tumor diameters, compared with sgCtrl mice ([96]figure 2D,E). Moreover, Pcna positive cells were significantly reduced in the sgPofut1 and shPofut1 tumors compared with the control group ([97]figure 2F and [98]online supplemental figure S2C). Thus, these findings demonstrate the crucial role of POFUT1 in promoting HCC growth in vivo. Figure 2. POFUT1 knockdown inhibits HCC progression and improves tumor immunosuppressive microenvironment. (A–B) Representative tumor images, (B) tumor weight and tumor volume of subcutaneous Hepa1-6 tumors (n=6). Scale bar, 1 cm. Data presented is the median with the range from six biological replicates. (C–E) Schematic outline showing sgCtrl and sgPofut1 MYC/Trp53^−/− HCC models by hydrodynamic plasmids injection. (D) Representative images of livers (n=6) from the indicated de novo mouse liver cancer models. (E) Tumor number per liver and max tumor diameter were measured. Scale bar, 1 cm. (F) Representative images of H&E, immunohistochemical Pofut1 and Pcna staining in the tumor tissues of mouse livers shown in (A) and (C). Scale bar, 50 µm. (G) Gating strategies for analysis of the tumor immune microenvironment by flow cytometry. (H) Relative proportions of lymphoid cells (CD45^+), T cells (CD45^+CD3^+), CD8^+ T cells (CD45^+CD3^+CD8^+) and CD4^+ T cells (CD45^+CD3^+CD4^+) in tumor tissues were analyzed by flow cytometry (n=5). (I) Numbers of lymphoid cells (CD45^+), T cells (CD45^+CD3^+), CD8^+ T cells (CD45^+CD3^+CD8^+) and CD4^+ T cells (CD45^+CD3^+CD4^+) per mg of tumor weight (cells per mg) (n=5). (J) Gating strategies for analysis of cytotoxic function of CD8^+ T cells by flow cytometry. (K) Relative proportions of IFN-G^+ and TNFA^+ of CD8^+ T cells in tumor tissues were analyzed by flow cytometry (n=5). (L) PD-1 expression on CD8^+ T cells in tumor tissues was analyzed by flow cytometry (n=5). Data represent mean±SD. P values were calculated using Student’s t-test. *p<0.05; **p<0.01; ***p<0.001; ****p<0.0001. HCC, hepatocellular carcinoma; IFN, interferon; MFI, median fluorescence intensity; PD-1, programmed death receptor 1; POFUT1, protein O-fucosyltransferase 1; TNF, tumor necrosis factor. [99]Figure 2 [100]Open in a new tab To assess the effects of POFUT1 on the immune microenvironment of HCC, mouse liver tumor tissues were collected for flow cytometry assay to characterize the impact of POFUT1 on different types of immune cells ([101]figure 2G, [102]online supplemental figure S2D,E). We found that the proportions and numbers of CD45^+ cells (lymphoid cells), CD45^+ CD3^+ cells (T cells), CD8^+ T cells and CD4^+ T cells were significantly increased in the sgPofut1 mouse group ([103]figure 2H and I). There were no differences in the proportions of natural killer (NK) and natural killer T (NKT) cells ([104]online supplemental figure S2F). We further examined the characteristics of CD8^+ T cells, and found that the cytotoxic function of CD8^+ T cells was enhanced in sgPofut1 group, as evidenced by elevated production of cytotoxic molecules IFN-G and tumor necrosis factor alpha (TNFA) in the tumor-infiltrating CD8^+ T cells ([105]figure 2J and K). Moreover, the PD-1 expression on CD8^+ T cells was lower in sgPofut1 group than in sgCtrl group ([106]figure 2L and [107]online supplemental figure S2G). In addition, immunofluorescence assays showed that the infiltration of CD8^+ T cells into the tumor tissues was increased in sgPofut1 group ([108]online supplemental figure S2H). Collectively, these results suggest that POFUT1 downregulation inhibits HCC progression by increasing immune cell infiltration and inhibiting CD8^+ T-cell exhaustion. POFUT1 overexpression promotes HCC progression and exacerbates immunosuppression To further investigate the role of POFUT1 gain-of-function in vivo, we interrogate the effect of POFUT1 overexpression in MYC/Trp53^−/− HCC model. POFUT1 gene was cloned into PT3-EF1A-MYC-IRES-luciferase by replacing the luciferase sequence, referred to as MYC-POFUT1 ([109]figure 3A). Indeed, POFUT1 overexpression mouse liver tumor group exhibited higher liver weight, liver/body weight ratio, tumor number, max tumor diameter and tumor burden ([110]figure 3B–3D and [111]online supplemental figure S3A). Moreover, Pcna positive cells were also significantly increased in MYC-POFUT1 tumors ([112]online supplemental figure S3B). In addition, we also harvested spleens from mice and the results showed that spleen weight and spleen/body weight ratio in MYC-POFUT1 group were higher than those in MYC group ([113]online supplemental figure S3C). These findings suggest that overexpression of POFUT1 facilitates HCC growth in vivo by regulating the immune microenvironment. Figure 3. POFUT1 promotes HCC development and exacerbates immunosuppression. (A–D) Schematic outline showing MYC and MYC-POFUT1 MYC/Trp53^–/– HCC models by hydrodynamic plasmids injection. (B) Representative images of livers (n=8) from the indicated de novo mouse liver cancer models. (C) Liver weight, liver/body weight ratio, (D) tumor number per liver, max tumor diameter and tumor burden determined by the ratio of tumor area/liver area were measured. Scale bar, 1 cm. (E) Representative images of H&E, immunohistochemical Pofut1 and Pcna staining in the tumor tissues of mouse livers shown in (A). Scale bar, 50 µm. (F) Gating strategies for analysis of the tumor immune microenvironment by flow cytometry. (G) Relative proportions of lymphoid cells (CD45^+), T cells (CD45^+CD3^+), CD8^+ T cells (CD45^+CD3^+CD8^+) and CD4^+ T cells (CD45^+CD3^+CD4^+) in tumor tissues were analyzed by flow cytometry (n=5). (H) Numbers of lymphoid cells (CD45^+), T cells (CD45^+CD3^+), CD8^+ T cells (CD45^+CD3^+CD8^+) and CD4^+ T cells (CD45^+CD3^+CD4^+) per mg of tumor weight (cells per mg) (n=5). (I) Relative proportions of IFN-G^+ and TNFA^+ of CD8^+ T cells in tumor tissues were analyzed by flow cytometry (n=5). (J) PD-1 expression on CD8^+ T cells in tumor tissues was analyzed by flow cytometry (n=5). (K) Gene Set Enrichment Analysis of signature of genes upregulated in comparison of naive CD8 T cells versus PD-1 high CD8 T cells in MYC-POFUT1 and MYC groups in RNA sequencing. The gene set “NAÏVE_VS_PD1HIGH_CD8_TCELL_UP” is available in the C7 IMMUNESIGDB collection (GSE26495_NAIVE_VS_PD1HIGH_CD8_TCELL_UP) at [114]http://www.msigdb.org. Data represent mean±SD. P values were calculated using Student’s t-test. *p<0.05; **p<0.01; ***p<0.001; ****p<0.0001. FDR, false discovery rate; HCC, hepatocellular carcinoma; IFN, interferon; MFI, median fluorescence intensity; NES, normalized enrichment score; PD-1, programmed death receptor 1; POFUT1, protein O-fucosyltransferase 1; TNFA, tumor necrosis factor alpha. [115]Figure 3 [116]Open in a new tab Next, we collected liver tumors to revalidate the impact of POFUT1 on different types of immune cells ([117]figure 3F and [118]online supplemental figure S3D). The flow cytometry analysis also showed that the proportions and numbers of CD45^+ cells, T cells, CD8^+ T cells and CD4^+ T cells in the MYC-POFUT1 group were lower than those in the MYC group ([119]figure 3G,H). There were no differences in the proportions of NK and NKT cells ([120]online supplemental figure S3E). Furthermore, we found that the production of cytotoxic molecules IFN-G and TNFA was decreased in the tumor-infiltrating CD8^+ T cells of MYC-POFUT1 group ([121]figure 3I). Moreover, the PD-1 expression on CD8^+ T cells was higher in MYC-POFUT1 group than in MYC group ([122]figure 3J and [123]online supplemental figure S3F). We also observed overexpression of POFUT1 resulted in a significant decrease in CD8^+ T cells infiltration ([124]online supplemental figure S3G). Then we performed RNA-seq on liver tumor tissues from the MYC-POFUT1 and MYC groups, and GSEA analysis also revealed that the naive_versus_PD-1 high_CD8_T cell_upregulated gene set was significantly enriched in the MYC group ([125]figure 3K), and the IFN response signaling gene sets were similarly enriched in the MYC group ([126]online supplemental figure S3H). Together, these results suggest that POFUT1 promotes HCC progression and immune evasion. POFUT1 interacts with PD-L1 to enhance its protein stability by inhibiting its ubiquitination and degradation We analyzed RNA-seq data for differentially expressed gene analysis between the two groups (MYC-POFUT1 vs MYC) to explore the mechanism underlying POFUT1-mediated HCC immune evasion. We identified 180 differentially expressed genes using the intersections between the differentially expressed and immune-related genes in a Venn diagram. Then we performed the Kyoto Encyclopedia of Genes and Genomes (KEGG) pathway enrichment analysis of these genes and the most significantly enriched pathway related to immunity was the T-cell receptor (TCR) signaling pathway ([127]figure 4A). GSEA also revealed that the TCR signaling pathway was significantly enriched in the MYC group ([128]figure 4B). As described above, POFUT1 could upregulate the expression of PD-1 on CD8^+ T cells in HCC and since the binding of PD-1 to its ligand, PD-L1, could inhibit TCR signaling pathway,[129]^9 37 we wondered whether POFUT1 inhibits TCR signaling pathway by upregulating PD-L1 expression in liver tumor cells. As expected, overexpression of POFUT1 could upregulate PD-L1 protein level in the Hepa1-6, Huh7 and non-HCC HEK293T cells ([130]figure 4C and [131]online supplemental figure S4A). Conversely, the knockdown of POFUT1 could downregulate the protein level of PD-L1 in the Hepa1-6, Huh7 and HEK293T cells ([132]figure 4D and [133]online supplemental figure S4B). Given the critical role of membrane PD-L1 in tumor immune escape,[134]^10 we further detected the PD-L1 level of cytomembrane using flow cytometry. We found that Pofut1 knockdown resulted in the downregulation of PD-L1, with a more significant decrease observed following IFN-G-induced PD-L1 expression ([135]figure 4E and [136]online supplemental figure S4C). Moreover, we also found the upregulation of membrane PD-L1 level in Hepa1-6 cells after overexpression of POFUT1 ([137]online supplemental figure S4D). In addition, we detected the expression level of PD-L1 in vivo by flow cytometry, and found tumor cells in the sgPofut1 group showed lower PD-L1 expression level, while those in the MYC-POFUT1 group exhibited higher PD-L1 expression ([138]figure 4F and [139]online supplemental figure S4E). Similarly, we examined the PD-L1 level in the liver tumor tissues from our animal models by IHC and found POFUT1 protein level showed a positive correlation with PD-L1 protein level ([140]online supplemental figure S4F-H). These results demonstrate that POFUT1 could upregulate PD-L1 protein expression in HCC cells and liver tumor tissues. To further verify the relationship between POFUT1 and PD-L1 expression in human HCC tissues, IHC staining was performed on 90 HCC samples to analyze the protein levels of POFUT1 and PD-L1. In HCC tissues, POFUT1 protein level showed a positive correlation with PD-L1 protein level ([141]figure 4G and [142]online supplemental figure S4I). These results indicate the clinical relevance of POFUT1 and PD-L1 expression. Figure 4. POFUT1 interacts with PD-L1 to enhance its protein stability. (A) Venn diagram depicting the overlap of genes containing immune genes and the differential expressed genes (DEG) in MYC-POFUT1 versus MYC in RNA-seq (left). Common genes for KEGG pathway analysis (right). The gene set “immune-gene” is available at [143]https://www.immport.org/home. (B) Gene Set Enrichment Analysis of T-cell receptor signaling pathway in MYC-POFUT1 and MYC groups in RNA-seq. (C) Western blot analysis of the levels of PD-L1 and POFUT1 in control and POFUT1 overexpression Hepa1-6 and Huh7 cells. (D) Western blot analysis of the levels of PD-L1 and POFUT1 in control and POFUT1 knockdown Hepa1-6 and Huh7 cells. (E) Expression level of PD-L1 on the cell membrane in Hepa1-6 cells with Pofut1 knockdown, with or without 24 hours incubation with IFN-G (n=3). (F) Expression level of PD-L1 on the cell membrane in tumor cells in vivo by flow cytometry (n=5). (G) The correlation between PD-L1 and POFUT1 IHC scores by correlation coefficient analysis in human HCC microarray (n=89). The Pearson coefficient was used to evaluate correlations. (H) Western blot analysis of PD-L1 protein in control and Pofut1 knockdown Hepa1-6 cells (top) and in control and POFUT1 overexpression Huh7 cells (bottom) treated with cycloheximide (CHX, 100 µg/mL). (I) Co-IP assay analyzed the interaction of endogenous PD-L1 with heterogenous expressed POFUT1-FLAG in HEK293T cells. (J) Co-IP assay analyzed the interaction of endogenous POFUT1 with heterogenous expressed PD-L1-FLAG in HEK293T cells. (K) In vitro GST pulldown assay analysis of the interaction of GST-PD-L1 and POFUT1. Black arrows indicated the Comassie blue staining of GST and GST-PD-L1. (L) Immunofluorescence assay showed PD-L1 co-localized with POFUT1 in the liver tumor tissue. Scale bar, 5 µm. (M) Western blot analysis of the levels of PD-L1 and POFUT1 in control, POFUT1-WT and POFUT1-MUT Hepa1-6 and Huh7 cells. (N–P) Ubiquitination assay of PD-L1 in HEK293T cells transfected with the indicated plasmids. Ubiquitinated PD-L1 was immunoprecipitated and subjected to western blot analysis with an antibody against ubiquitin. For E and F data represent mean±SD. P values were calculated using Student’s t-test. **p<0.01; ***p<0.001; ****p<0.0001. Co-IP, co-immunoprecipitation; FDR, false discovery rate; HCC, hepatocellular carcinoma; IFN, interferon; KEGG, kyoto encyclopedia of genes and genomes; MUT, mutant; NES, normalized enrichment score; PD-L1, programmed death ligand 1; POFUT1, protein O-fucosyltransferase 1; RNA-seq, RNA sequencing; WT, wild-type. [144]Figure 4 [145]Open in a new tab The upregulation of PD-L1 protein level may result from the activation of PD-L1 transcription and/or enhanced PD-L1 protein stability. First, we examined the mRNA level of PD-L1 in Hepa1-6 cells; however, the quantitative PCR showed that POFUT1 did not activate PD-L1 transcription ([146]online supplemental figure S4J). Next, we wondered whether POFUT1 could enhance the stability of PD-L1 protein, the stability of PD-L1 was explored using the translation inhibitor cycloheximide, and we found that knockdown of POFUT1 could shorten the half-life of PD-L1 protein in the Hepa1-6 cells, moreover, ectopic expression of POFUT1 had an opposite result in the Huh7 cells ([147]figure 4H, [148]online supplemental figure S4K,L). These results indicate that POFUT1 enhances the stability of PD-L1 protein. Next, we wondered whether POFUT1 might interact with PD-L1 to promote its stability. To this end, using the co-immunoprecipitation (Co-IP) assays, the interaction of endogenous PD-L1 with FLAG-POFUT1 in HEK293T cells was verified ([149]figure 4I). Similarly, the interaction of endogenous POFUT1 with FLAG-PD-L1 was also found in HEK293T cells ([150]figure 4J). Furthermore, GST pulldown assays also indicated that POFUT1 interacted with PD-L1 ([151]figure 4K). Moreover, immunofluorescence assay revealed that endogenous PD-L1 co-localized with POFUT1 in the liver tumor tissue ([152]figure 4L) and Huh7 cells ([153]online supplemental figure S4M). Given POFUT1 is a key enzyme in O-fucose biosynthesis and O-fucosylation is a type of protein post-translational modification, we investigated the role of its enzymatic activity in PD-L1 upregulation. We first constructed POFUT1 wild-type (POFUT1-WT) plasmid and POFUT1 mutant (POFUT1-MUT) plasmid with Arg240 changed to Ala, which lost its enzyme activity.[154]^38 Western blot analysis showed that ectopic expression of POFUT1-MUT still could upregulate PD-L1 protein level in Hepa1-6, Huh7 and HEK293T cells ([155]figure 4M and [156]online supplemental figure S4N). In addition, we further detected the level of PD-L1 on the cell membrane and found a similar result in Hepa1-6 cells ([157]online supplemental figure S4O). These results demonstrate that POFUT1 promotes PD-L1 protein stability independently of its protein-O-fucosyltransferase activity. Given that the degradation of PD-L1 protein mainly involves ubiquitin proteasome or lysosome pathways,[158]^10 39 we wondered whether POFUT1 promotes the stability of PD-L1 protein by inhibiting its ubiquitination. To test this hypothesis, Co-IP assays were conducted using the anti-FLAG monoclonal antibody (mAb), followed by the detection of the ubiquitin-conjugated FLAG-PD-L1. Overexpression of POFUT1 markedly reduced the level of the ubiquitin-conjugated FLAG-PD-L1 ([159]figure 4N). Conversely, knockdown of POFUT1 could increase the level of ubiquitin-conjugated FLAG-PD-L1 ([160]figure 4O). In addition, we further confirmed that the role of POFUT1 inhibit the ubiquitination of PD-L1 protein independently of its protein-O-fucosyltransferase activity ([161]figure 4P). These results showed that POFUT1 regulates PD-L1 expression via the ubiquitination pathway. Together, these data suggest that POFUT1 upregulates the stability of PD-L1 by inhibiting its ubiquitination and protein degradation. POFUT1 upregulates PD-L1 expression via preventing TRIM21-mediated PD-L1 ubiquitination Given that POFUT1 promotes the protein stability of PD-L1 by inhibiting its ubiquitination, we wondered whether POFUT1 could impair some E3 ligase-mediated PD-L1 ubiquitination and degradation. To identify the potential E3 ligases, Co-IP and liquid chromatography/tandem mass spectrometry were conducted to identify the potential binding proteins of POFUT1 ([162]figure 5A and [163]online supplemental file 2). Through mass spectrometry, six E3 ubiquitin ligase proteins were associated with POFUT1 ([164]figure 5B). The top-enriched E3 ligase, UBR5, has been reported to regulate PD-L1 at the transcriptional level, and this process is independent of its E3 ligase activity.[165]^40 Besides UBR5, the E3 ubiquitin ligase with the most significant p value was TRIM21 ([166]figure 5C). Although previous study has reported that TRIM21 may be involved in regulating the protein level of PD-L1 in lung cancer,[167]^41 however, it is unclear whether TRIM21 can interact with PD-L1 to promote its ubiquitination degradation. Hence, we wondered whether POFUT1 stabilizes PD-L1 via downregulating TRIM21-mediated PD-L1 ubiquitination. To verify this hypothesis, we first explored whether TRIM21 could reduce the protein level of PD-L1. Indeed, overexpression of TRIM21 could downregulate PD-L1 expression in Huh7 and HEK293T cells, importantly, the effect of TRIM21 could be almost abolished on POFUT1 overexpression ([168]figure 5D,E). Next, we further investigated whether TRIM21 could promote the ubiquitination of PD-L1. Co-IP assays were conducted to detect the level of the ubiquitin-conjugated FLAG-PD-L1. TRIM21 overexpression indeed could increase the ubiquitin level of PD-L1, similarly, POFUT1 could abolish the effect of TRIM21-mediated PD-L1 ubiquitination ([169]figure 5F). As an E3 ligase, TRIM21 always targets substrates by protein interacting, we further examined the interaction between TRIM21 with PD-L1. As expected, Co-IP assays revealed that TRIM21 could interact with PD-L1 in HEK293T cells ([170]figure 5G,H). Similarly, the interaction of TRIM21 with POFUT1 in HEK293T cells was also verified ([171]figure 5I,J). Furthermore, immunofluorescence assay revealed that endogenous TRIM21 co-localized with POFUT1 and PD-L1 in the Huh7 cells ([172]figure 5K). Finally, we further explored whether POFUT1-mediated the stability of PD-L1 protein by preventing the interaction between TRIM21 and PD-L1. As expected, Co-IP assays demonstrated that POFUT1 indeed could downregulate the binding of PD-L1 and TRIM21 in HEK293T cells ([173]figure 5L). These results suggest that POFUT1 can reduce the interaction between PD-L1 and TRIM21 to inhibit the TRIM21-mediated PD-L1 ubiquitination. Figure 5. POFUT1 inhibits TRIM21-mediated PD-L1 ubiquitination and degradation. (A–B) Identify potential substrates of POFUT1 related to E3 ubiquitin ligases by liquid chromatography-tandem mass spectrometry. (C) The TRIM21 peptides identified through mass spectrometry were shown. (D–F) Western blot analysis demonstrated that PD-L1 decrease and (F) ubiquitination increase induced by TRIM21 overexpression were abolished by overexpression of POFUT1 in Huh7 cells and HEK293T cells. (G–H) Co-IP assay analyzes the interaction of TRIM21 with PD-L1 in HEK293T cells. (I–J) Co-IP assay analyzes the interaction of TRIM21 with POFUT1 in HEK293T cells. (K) Immunofluorescence assay shows TRIM21 co-localized with POFUT1 and PD-L1 in Huh7 cells. Scale bar, 10 µm. (L) Western blot analysis demonstrated that POFUT1 overexpression weakened the binding of TRIM21 to PD-L1 in HEK293T cells. Co-IP,co-immunoprecipitation; PD-L1, programmed death ligand 1; POFUT1, protein O-fucosyltransferase 1; TRIM21, tripartite motif containing 21. [174]Figure 5 [175]Open in a new tab PD-L1 is required for the tumor-promoting effect of POFUT1 in HCC Next, we tried to figure out the contribution of PD-L1 to the tumor-promoting effect of POFUT1 in HCC. First, we overexpressed PD-L1 in the de novo MYC/Trp53^−/− HCC model with or without POFUT1 and found that phenotypes of the sgPofut1 mice group were almost rescued by PD-L1 overexpression ([176]figure 6A–C). Then, we further investigated whether POFUT1 indeed cooperates with PD-L1 to promote HCC progression. To test this hypothesis, we knocked out Pdl1 in the de novo MYC/Trp53^−/− HCC model with control or POFUT1 overexpression, and found that the tumor-promoting phenotype of POFUT1 overexpression in HCC was almost abolished on Pdl1 depletion ([177]figure 6D–F and [178]online supplemental figure S5A). These results indicate that the tumor-promoting effect of POFUT1 on HCC progression requires PD-L1 expression. In addition, we further examined the functional promotion of HCC by POFUT1 whether entirely relies on PD-L1. To this end, we constructed the Hepa1-6 shPofut1 Pdl1^−/− cells. And the level of Pdl1 protein was determined by western blot ([179]online supplemental figure S5B). The experimental results revealed that knocking down Pofut1 in combination with Pdl1 knockout further suppressed tumor progression compared with the Pdl1 knockout ([180]online supplemental figure S5C,D). These findings suggest that the promotion of tumor progression by POFUT1 partially relies on PD-L1. Figure 6. PD-L1 is required for the immune evasion-promoting effect of POFUT1 in hepatocellular carcinoma. (A–C) The representative images of livers from de novo mouse liver cancer models after hydrodynamic plasmids injection. (B) Tumor number per liver, tumor number (>1 mm) per liver and (C) tumor burden determined by the ratio of tumor area/liver area were measured (n=11). Scale bar, 1 cm. (D–F) The representative images of livers from de novo mouse liver cancer models after hydrodynamic plasmids injection. (E) Tumor number per liver, tumor number (>1 mm) per liver and (F) tumor burden determined by the ratio of tumor area/liver area were measured (n=6). Scale bar, 1 cm. (G) Representative images of IHC CD8, GZMB and IFN-G and numbers of their positive cells (cells/mm^2) (n=4) in the tumor tissues of mouse livers shown in (A). Scale bar, 50 µm. (H) Representative images of IHC CD8, GZMB and IFN-G and numbers of their positive cells (cells/mm^2) (n=4) in the tumor tissues of mouse livers shown in (D). Scale bar, 50 µm. Data represent mean±SD. P values were calculated using one-way analysis of variance. ns, no significance; *p<0.05; **p<0.01; ***p<0.001; ****p<0.0001. IFN, interferon; IHC, immunohistochemical; PD-L1, programmed death ligand 1; POFUT1, protein O-fucosyltransferase 1. [181]Figure 6 [182]Open in a new tab Then, we investigated the tumor immune microenvironment of the de novo MYC/Trp53^−/− HCC mouse model by evaluating the T cell (CD3) and CD8^+ T cell (CD8) infiltration and levels of T-cell cytotoxic markers (GZMB and IFN-G). IHC analysis showed that the improvement of the immune microenvironment of the sgPofut1 group could be abolished by PD-L1 overexpression ([183]figure 6G and [184]online supplemental figure S5E). Conversely, the immunosuppressive status of the MYC-POFUT1 group could be rescued by Pdl1 depletion ([185]figure 6H and [186]online supplemental figure S5F). These results demonstrate that the immunosuppressive function of POFUT1 in HCC is dependent on PD-L1. Given the CD8^+ T-cell subset may play a vital role in the antitumor effect of POFUT1 knockdown, we further verify whether the POFUT1 knockdown-mediated antitumor effect is dependent on CD8^+ T cells, we used an anti-CD8α antibody to block the CD8^+ T-cell activity in C57BL/6 mice. In mice bearing Pofut1 knockdown tumors, anti-CD8α antibody treatment significantly promoted the tumor growth and the antitumor effect of POFUT1 knockdown was almost abolished ([187]online supplemental figure S5G,H). These results suggest that the role of POFUT1 in immune evasion may mainly depend on CD8^+ T cells. Inhibition of POFUT1 in HCC enhances the efficacy of anti-PD-1 therapy Given that POFUT1-mediated PD-L1 stability promotes HCC progression and tumor immunosuppression, we sought to determine whether POFUT1 could predict immunotherapy response and might be a potential target to improve immunotherapy efficacy. We first analyzed immunotherapy responses between the POFUT1 and control groups using our RNA-seq data, and GSEA revealed that the MYC-POFUT1 group was associated with poor prognosis after ICB treatment ([188]figure 7A). Considering that the cytotoxic T lymphocyte (CTL) level can affect ICB efficacy,[189]^42 we next examined the relationship between POFUT1 and cytotoxic levels. We found that POFUT1 was negatively associated with cytotoxic markers (GZMA, GZMB, NKG7, GNLY and CST7) in TCGA_LIHC and CHCC_HBV data sets ([190]online supplemental figure S6A,B). Moreover, the results showed that expression of POFUT1 negatively correlated with CTL level in TCGA_LIHC ([191]figure 7B). The similar results were also found in the melanoma and bladder cancer data sets on ICB therapy ([192]figure 7C and [193]online supplemental figure S6C). Next, we wondered whether POFUT1 could affect CTL-mediated ICB effectiveness. As expected, the high CTL group displayed better survival in the patients with low POFUT1 expression in the melanoma data set on anti-PD-1 therapy ([194]figure 7D). Moreover, we further found the expression of POFUT1 was increased in the melanoma and pancancer cohorts that did not respond to anti-PD-1 therapy (non-response group) ([195]figure 7E and [196]online supplemental figure S6D). Then, we explored the relationship between POFUT1 expression and immunotherapy response in the anti-PD-1 treatment cohorts. We found patients with POFUT1 low expression had better survival outcomes in melanoma and pancancer data sets ([197]figure 7F and [198]online supplemental figure S6E). Figure 7. Inhibition of POFUT1 in hepatocellular carcinoma can synergize with anti-PD-1 therapy. (A) Gene Set Enrichment Analysis of the immunotherapy prognosis-related pathways in MYC-POFUT1 and MYC groups in RNA sequencing. (B–C) Correlation analysis of the cytotoxic T lymphocyte (CTL) level and the expression of POFUT1 in different cancers using the TIDE website ([199]http://tide.dfci.harvard.edu/). (D) The association between the CTL level and progression-free survival (PFS) in POFUT1 high and low groups in melanoma using the TIDE website ([200]http://tide.dfci.harvard.edu/). (E) The expression analysis of POFUT1 in non-response (NR) and response (R) groups of immunotherapy in melanoma using the ROC Plotter website ([201]https://www.rocplot.com/). (F) Survival analysis of overall survival and PFS of POFUT1 levels in melanoma patients receiving immunotherapy using KM plotter website ([202]https://kmplot.com/analysis/index.php?p=background). (G) Schematic depiction of the tumor formation in C57BL/6 mice. Mice (n=6) were injected with control or shPofut1 Hepa1-6 cells and further treated with anti-mouse PD-1 antibodies. Anti-mouse PD-1 antibodies were intraperitoneally injected (100 µg per mouse) once every 3 days for four times. (H–K) Representative tumor images, (I) tumor weight, (J) tumor volume and (K) growth curves of tumor volume measured at indicated time points for subcutaneous Hepa1-6 tumors (n=6). Scale bar, 1 cm. (L–M) Representative images of immunohistochemical CD8, IFN-G, GZMA and GZMB and numbers of their positive cells (cells/mm^2) (n=4) in the tumor tissues of mouse livers shown in (H). Scale bar, 50 µm. For I and J data presented is the median with the range from six biological replicates. For K and M data represent mean±SD. For I J and M p values were calculated using one-way ANOVA. For K p values were calculated using two-way ANOVA. *p<0.05; **p<0.01; ****p<0.0001. ANOVA, analysis of variance; FDR, false discovery rate CTLA-4, cytotoxic T-lymphocyte associated protein-4; IFN, interferon; NES, normalized enrichment score; PD-1, programmed death receptor 1; POFUT1, protein O-fucosyltransferase 1; TCGA_LIHC, The Cancer Genome Atlas Liver Hepatocellular Carcinoma. [203]Figure 7 [204]Open in a new tab Next, we examined the possibility of POFUT1 inhibition in sensitizing anti-PD-1 therapy in HCC. We established a subcutaneous implantation mouse model using Hepa1-6-SCR or Hepa1-6-shPofut1 cells and divided them into two groups that were administered IgG and PD-1 antibodies, respectively ([205]figure 7G). The growth of tumor volume was monitored and tumors were harvested and measured after 2 weeks. The results revealed that inhibition of POFUT1 expression not only prevented HCC progression, but also sensitized liver tumor cells to anti-PD-1 therapy ([206]figure 7H–7K). Furthermore, IHC analysis showed that Pofut1 knockdown combined with anti-PD-1 treatment significantly increased T cell and CD8^+ T-cell infiltration and levels of T-cell cytotoxic markers IFN-G, GZMA, and GZMB ([207]figure 7L,M and [208]online supplemental figure S6F). Taken together, these results indicate that POFUT1 may be a potential marker for predicting ICB treatment response and inhibiting POFUT1 expression can synergize with anti-PD-1 treatment to restore T-cell function and inhibit tumor progression. Discussion While PD-1/PD-L1 blockade immunotherapy has been employed in various human cancer types, including HCC, patients with advanced HCC often exhibit a limited response rate to anti-PD-1 monotherapy. Recently, two clinical trials of patients with HCC used anti-PD-1 therapy did not reach a predetermined endpoint.[209]^43 Thus, optimal patient selection schemes and combination treatment strategies remain major challenges in the ICB therapy field. In this study, we demonstrated for the first time that POFUT1 may be a potential marker for predicting ICB treatment response and inhibiting POFUT1 expression in HCC can synergize with anti-PD-1 treatment. Our findings revealed that inhibition of POFUT1 may be a potential therapeutic strategy to enhance the efficacy of immune therapy in HCC. Thus, it is of great importance to develop therapeutic approaches that target POFUT1 concurrently with ICB therapy for HCC immunotherapy. Our study also revealed that POFUT1 functions as a crucial regulator of liver cancer immune evasion. Although POFUT1 is highly expressed in cancers and promotes tumor progression, its role of POFUT1 in cancer immunity is largely unexplored, especially in HCC. Our study is the first to report the regulatory effect of POFUT1 on the tumor microenvironment. In this study, we identified POFUT1, as an important factor, that promotes HCC progression and immunosuppressive microenvironment by reducing immune cell infiltration and promoting CD8^+ T-cell exhaustion, finally facilitating tumor immune evasion. Mechanistically, we demonstrated that POFUT1 upregulates PD-L1 at the protein level and enhances its stability by suppressing TRIM21-mediated ubiquitination and protein degradation, resulting in the upregulation of PD-L1 protein in HCC cells. Therefore, our current work demonstrates POFUT1 as a novel tumor immune evasion regulator and may serve as a potential therapeutic target against cancer immune evasion ([210]online supplemental figure S7). POFUT1, a fucosyltransferase resident in the endoplasmic reticulum, is responsible for fucosylating the EGF-like domains, playing a key role in the modification of Notch receptors and their ligands.[211]^44 Notch receptors require POFUT1 to add O-fucose to conserved Ser or Thr residues in the EGF-like repeats of its extracellular domain for signaling through delta-like and Jagged ligands and the Notch signaling pathway is a classical oncogenic signaling pathway.[212]^45 46 Previous studies have mainly reported that POFUT1 regulates the Notch pathway to promote tumor progression.[213]^47 48 Our current study suggests that the tumor-promoting and immune evasion effects of POFUT1 require PD-L1, at least in HCC. The POFUT1-PD-L1 axis is likely to play a key role in POFUT1 regulating immune evasion. In this study, we also found that concomitant loss of POFUT1 and PD-L1 could further slow the liver tumor progression compared with PD-L1 single knockout group, which suggests that the promotion role of POFUT1 in HCC progression may also regulate other factors that contribute to its promotion of tumor progression, not entirely dependent on PD-L1 expression. Our finding further revealed that POFUT1 promotes PD-L1 protein stability independent of its protein-O-fucosyltransferase activity. Consistently, previous studies have shown that PD-L1 is mainly extensively modified by N glycosylation, while O-glycosylation has no obvious effect on it.[214]^10 49 Thus, our findings demonstrate that POFUT1-mediated PD-L1 protein stability promotes HCC progression and extends the understanding of PD-L1 protein dysregulation in cancer cells. In summary, our present study demonstrates that POFUT1-mediated PD-L1 protein stability is crucial for promoting HCC progression and immune evasion, thereby establishing a direct regulatory mechanism that links these tumor immune evasion regulators. Consistent with this role, POFUT1 high expression in cancer shows therapeutic insensitivity to ICB therapies. Similarly, genetic knockdown of POFUT1 expression in HCC can synergize with anti-PD-1 therapy. Therapeutically, inhibition of POFUT1 may be a potential novel strategy to enhance the efficacy of anti-PD-1 therapy in HCC. supplementary material online supplemental file 1 [215]jitc-12-6-s001.docx^ (4.7MB, docx) DOI: 10.1136/jitc-2024-008917 online supplemental file 2 [216]jitc-12-6-s002.pdf^ (1.6MB, pdf) DOI: 10.1136/jitc-2024-008917 Acknowledgements