Targeting MAN1B1 potently enhances bladder cancer antitumor immunity via deglycosylation of CD47

Abstract

Background: Only a few bladder cancer patients benefit from anti-programmed cell death protein 1/programmed cell death ligand 1 immunotherapy. The cluster of differentiation 47 (CD47) plays an important role in tumor immune evasion. CD47 is a highly glycosylated protein, however, the mechanisms governing CD47 glycosylation and its potential role in immunosuppression are unclear. Therefore, this study aimed to evaluate the function of CD47 glycosylation in bladder cancer.

Methods: Western blotting, immunohistochemistry, and flow cytometry were used to measure protein expression, protein-protein interactions, and phagocytosis in bladder cancer. A murine model was employed to investigate the impact of mannosidase alpha class 1B member 1 (MAN1B1) modification of CD47 on anti-phagocytosis in vivo. An ex vivo model, patient-derived tumor-like cell clusters, was used to examine the effect of targeting MAN1B1 on phagocytosis.

Results: Our research identified that aberrant CD47 glycosylation was responsible for its immunosuppression. The glycosyltransferase MAN1B1 responsible for CD47 glycosylation was highly expressed in bladder cancer. Abnormal activation of extracellular signal-regulated kinase (ERK) was significantly associated with MAN1B1 stability by regulating the interaction between MAN1B1 and the E3 ubiquitin ligase HMG-CoA reductase degradation 1 (HRD1). Mechanistically, abnormally activated ERK stabilized MAN1B1, resulting in the glycosylation of CD47 and facilitating immune evasion by enhancing its interaction with signal-regulatory protein alpha (SIRP-α). In vitro and in vivo experiments demonstrated that MAN1B1 knockout weakened CD47-mediated anti-phagocytosis. MAN1B1 inhibitors promoted phagocytosis without causing anemia, offering a safe alternative to anti-CD47 therapy.

Conclusions: This comprehensive analysis uncovered that ERK activation stabilizes MAN1B1 by regulating the interaction between MAN1B1 and HRD1, facilitates immune evasion via CD47 glycosylation, and presents new potential targets and strategies for cancer immunotherapy that do not cause anemia.

Keywords: Bladder cancer; HMG‐CoA reductase degradation 1; cluster of differentiation 47; extracellular signal‐regulated kinase; immunotherapy; macrophage; mannosidase alpha class 1B member 1; phagocytosis.

FIGURE 1

The glycosylation of CD47 was critical for its interaction with SIRP‐α. (A) Cd47 was knocked out in the bladder cancer cell line MB49. (B‐D) Tumor growth of MB49 in mice (n = 4 mice per group). (B) Images of endpoint subcutaneous tumors. (C) Tumor volumes of MB49 in vivo. (D) Tumor weight of MB49 at endpoint. (E) Western blotting analysis of CD47 in normal and tumor tissues of bladder cancer. (F) Western blotting analysis of cell lysates from tumor tissues and 293T treated with PNGase F for 1 h at 37°C in vitro. (G) Glycoprotein staining and Coomassie blue staining of PNGase F treated purified CD47 (ECD)‐biotin. Horseradish peroxidase and soybean trypsin inhibitor served as positive and negative controls, respectively. (H) Schematic diagram of CD47 amino acid sequence alignment among different species. The five putative NXT motifs are highlighted in red. (I) Western blotting of the protein expression pattern of CD47 WT and mutants overexpressed in 293T cells. (J) Western blotting of the protein expression pattern of CD47‐WT and 5NQ treated with PNGase F for 1 h at 37°C in vitro. (K‐L) Analysis of CD47‐SIRP‐α binding by IP. (K) The lysates of 293T cells overexpressing FLAG‐tagged CD47 WT or 5NQ mutant were incubated with beads which had conjugated to SIRP‐α‐Fc and then observed by Western blotting with anti‐FLAG antibody. (L) The lysates of 293T cells overexpressing FLAG‐tagged SIRP‐α were incubated with beads which had conjugated to CD47‐WT or 5NQ‐Fc and then observed by Western blotting with anti‐FLAG antibody. (M) The lysates of 293T cells overexpressing CD47 WT or the indicated NQ mutants were incubated with beads which had conjugated to SIRP‐α‐Fc and then observed by Western blotting with anti‐FLAG antibody. (N‐O) Analysis of CD47 knockout in J82 and T24 cells by Western blotting. (P) CD47‐WT or 5NQ was overexpressed in CD47‐knocked out J82, T24 or 293T cells to detect the expression of CD47 and its affinity with SIRP‐α by flow cytometry. (Q‐R) Representative flow cytometry images (Q) and quantification (R) depicted the In vitro phagocytosis of J82 cells, which were knocked out CD47 and then stably rescued with either CD47‐WT or CD47‐5NQ. Boxes were representative of GFP⁺ CD11b⁺ macrophages. (S‐W) Tumor growth of J82 in NSG mice (n = 4 mice per group). (S) Images of endpoint subcutaneous tumors. (T) Tumor volumes of J82 in vivo. (U) Tumor weight of J82 at endpoint. (V‐W) Representative flow cytometry (V) and quantification (W) demonstrated TAM phagocytosis in GFP⁺ CD47‐WT group and GFP⁺ CD47‐5NQ group (n = 4). Numbers indicated frequency of phagocytosis events out of all TAMs after engraftment. Boxes were representative of GFP⁺ CD11b⁺ macrophages. P values in (C‐D, P‐R, T‐W) were determined by a two‐tailed unpaired Student's t‐test, error bars represent mean ± SD. NS, not significant; * P < 0.05, ** P <0.01, *** P <0.001. Abbreviations: CD47, cluster of differentiation 47; SIRP‐α, signal‐regulatory protein alpha; TAM, tumor‐associated macrophages; PNGase F, peptide‐N‐glycosidase F; GFP, green fluorescent protein; ECD, Extracellular domain; NXT motif, N‐glycosylation motif; WT, Wild type; 5NQ, 5 substitution of asparagine with glutamine mutant; NSG, NoD.Cg.Prkdcscidll2rgem1Smoc mice; gCD47, glycosylated CD47; ngCD47, nonglycosylated CD47; IP, immunoprecipitation; SD, standard deviation.

FIGURE 2

MAN1B1 was involved in the glycosylation process of CD47 and exhibited high expression levels in bladder cancer. (A) MS analysis result of CD47. Venn diagram was performed according to glycosyltransferases identified in CD47 MS analysis from 293T and J82 cells and well‐known glycosyltransferase. (B) Diagram of docking between CD47 and MAN1B1. The binding score was ‐726. (C) Analysis of interaction between CD47 and MAN1B1 by IP. (D) Expression levels of CD47 and MAN1B1 in 12 human BLCA fresh samples by Western blotting. N, normal matched tissue; T, tumor tissue. (E‐F) The band intensity was quantified and normalized to compare CD47 and MAN1B1 levels (n = 12). (G‐H) Images and statistical results of IHC staining of MAN1B1 in a tissue microarray (n = 47). (I) Analysis of MAN1B1 knockout in J82 and T24 cells by Western blotting. (J) Detected changes in LCA affinity of whole cell lysates under MAN1B1 knockout or control conditions in J82 and T24. (K) Survival analysis of MAN1B1 and CD47 in bladder cancer. P values in (E‐H) were determined by a two‐tailed unpaired Student's t‐test, error bars represent mean ± SD. NS, not significant; * P < 0.05, ** P <0.01, *** P <0.001. Abbreviations: CD47, cluster of differentiation 47; SIRP‐α, signal‐regulatory protein alpha; MAN1B1, mannosidase alpha class 1B member 1; IHC, Immunohistochemical staining; LCA, Lens culinaris agglutinin; MS, Mass spectrometry; IP, immunoprecipitation; BLCA, bladder urothelial carcinoma; B7H3, B7 homolog 3; SD, standard deviation.

FIGURE 3

Identification of MAN1B1 as a modulator of CD47‐SIRP‐α axis. (A‐B) Analysis of CD47‐SIRP‐α binding was measured in MAN1B1 knockout and control cells by IP. (A) The lysates of sgControl or sgMAN1B1 293T cells overexpressing FLAG‐tagged CD47 were incubated with beads which had conjugated to SIRP‐α‐Fc and then observed by Western blotting with anti‐FLAG antibody. (B) The whole cell lysates of sgControl or sgMAN1B1 T24 cells were incubated with beads which had conjugated to SIRP‐α‐Fc and then observed by Western blotting with anti‐CD47 antibody (left). The band intensity was quantified and normalized to exhibit changed affinity after MAN1B1 knockout (right). (C‐F) Flow cytometry plots of surface binding of anti‐human CD47 antibody clone B6H12 (anti‐hCD47) and SIRP‐α to T24 (C‐D) or J82 (E‐F) sgControl and sgMAN1B1 cells. Values indicated MFI relative to sgControl cells stained with the same reagent (D, F). (G) IF staining showed bound SIRP‐α protein (red) and DAPI‐stained nuclei (blue) in T24 cells. (H) Analysis of CD47‐SIRP‐α binding between normal and tumor tissues by IP. (I) The affinity intensity was quantified and normalized to show the association between the expression levels of MAN1B1 and the CD47‐SIRP‐α binding levels. (J‐M) Representative flow cytometry images (J, L) and quantification (K, M) demonstrated the phagocytic activity of BMDMs to both control and MAN1B1 knockout J82 cells (J‐K, n = 9) or T24 cells (L‐M, n = 4). Boxes were representative of GFP⁺ CD11b⁺ macrophages. (N‐Q) Representative flow cytometry images (N, P) and quantification (O, Q) demonstrated the phagocytosis of PBMCs to both control and MAN1B1 knockout J82 cells (N‐O, n = 4), as well as in control and MAN1B1 and CD47 knockout T24 cells (P‐Q, n = 3). Boxes were representative of GFP⁺ CD11b⁺ macrophages. (R) Human bladder cancer cells J82 were transplanted subcutaneously into NSG mice. (S‐Y) Tumor growth of J82 sgControl and sgMAN1B1 in mice (n = 4 mice per group). (S) Images of endpoint subcutaneous tumors. (T) Tumor grow curves of J82 cells in vivo. (U) Tumor weight of J82 cells at endpoint. (V) Representative flow cytometry plots demonstrated TAM phagocytosis in GFP⁺ control group (left) versus GFP⁺ MAN1B1 knockout group (right); numbers indicated the frequency of phagocytosis events out of all TAMs after engraftment. Boxes were representative of GFP⁺ CD11b⁺ macrophages. (W) Phagocytosis ratio was analyzed by statistics (n = 4). (X‐Y) Frequency of TAMs positive for CD80 (M1‐like) as per gating in vivo (n = 4). P values (A‐B, D, F, I‐Q and T‐Y) were determined by a two‐tailed unpaired Student's t‐test, error bars represent mean ± SD. NS, not significant; * P < 0.05, **P < 0.01, *** P < 0.001. Abbreviations: CD47, cluster of differentiation 47; SIRP‐α, signal‐regulatory protein alpha; MAN1B1, mannosidase alpha class 1B member 1; BMDMs, bone marrow‐derived macrophages; PBMCs, peripheral blood mononuclear cells; TAM, tumor‐associated macrophages; NSG, NoD.Cg.Prkdcscidll2rgem1Smoc mice; GFP, green fluorescent protein; MFI, mean fluorescence intensity; IF, immunofluorescence; IP, immunoprecipitation; BLCA, bladder urothelial carcinoma; B7H3, B7 homolog 3; SD, standard deviation.

FIGURE 4

The MAN1B1 inhibitor DHT I reduced the affinity between CD47 and SIRP‐α, promoted phagocytosis of bladder cancer cells via macrophage in vitro and in vivo. (A) Chemical structure of dihydrotanshinone I (DHT I). (B) Predicted three‐dimensional model of interaction binding between DHT I (ZINC ID: 8681480) and MAN1B1 (PDB: 1×9D) as shown by computational docking. Binding energy: ‐8.4 kcal/mol. (C) Detected changes in LCA affinity of whole cell lysates from J82, T24 or 293T cells treated with DHT I (0.625 µmol/L). (D) Flow cytometry plot of surface binding of anti‐human CD47 antibody clone B6H12 (anti‐hCD47) and SIRP‐α to J82 cells which were treated with DHT I (0.625 µmol/L). Values indicated MFI relative to control cells stained with the same reagent (bottom). (E) 293T cells overexpressing FLAG‐tagged CD47 were treated with DHT I for 24h, then the whole cell lysates were incubated with beads which had conjugated to SIRP‐α‐Fc and observed by Western blotting with anti‐FLAG antibody. (F) Representative flow cytometry image depicted the process of BMDMs mediated phagocytosis of J82 sgControl or sgMAN1B1 cells treated with or without DHT I for 24h in vitro. Boxes were representative of GFP⁺ CD11b⁺ macrophages. (G) Phagocytosis ratio was analyzed by statistics. (H) A schematic treatment plan for NSG mice bearing subcutaneous T24 tumors. Mice were subcutaneously implanted with T24 sgControl or sgMAN1B1 cells and treated with either control vehicle or DHT I, respectively. (I‐M) Tumor growth in DHT I‐treated mice (n = 4 mice per group). (I) Images of endpoint subcutaneous tumors. (J) Tumor weight of T24 tumors at endpoint. (K) Tumor volumes of T24 in vivo. (L‐M) Representative flow cytometry plots demonstrated TAM phagocytosis in T24 tumor, with numbers indicated the frequency of phagocytosis events out of all TAMs after treatment with DHT I. Boxes were representative of GFP⁺ CD11b⁺ macrophages. (N) A representative case of bladder cancer with PTCs culture was presented in the bright field view. (O) Representative flow cytometry image depicted phagocytosis in PTCs model treated with CD47 antibody or DHT I. Boxes were representative of KRT18⁺ CD11b⁺ macrophages. (P) Analysis of phagocytosis ratio after CD47 antibody or DHT I treatment in the PTCs model. P values in (D, G and J‐M) were determined by a two‐tailed unpaired Student's t‐test, error bars represent mean ± SD. NS, not significant; * P < 0.05, **P < 0.01, ***P < 0.001. Abbreviations: CD47, cluster of differentiation 47; SIRP‐α, signal‐regulatory protein alpha; MAN1B1, mannosidase alpha class 1B member 1; DHT I, dihydrotanshinone I; TAM, tumor‐associated macrophages; PTCs, patient‐derived tumor‐like cell clusters; DMSO, Dimethyl sulfoxide; GAPDH, Glyceraldehyde‐3‐phosphate dehydrogenase; LCA, Lens culinaris agglutinin; KRT18, Keratin 18; GFP, green fluorescent protein; MFI, mean fluorescence intensity; BMDMs, bone marrow‐derived macrophages; NSG, NoD.Cg.PrkdcscidIl2rgem1Smoc mice; SD, standard deviation.

FIGURE 5

The targeted therapy directed at MAN1B1 exhibited negligible adverse effects. (A) Analysis of erythrocytes, hemoglobin, and platelets of mice treated with DHT I. (B) Analysis of hepatorenal function of mice treated with DHT I. (C) Analysis of MAN1B1 expression levels in erythrocytes from healthy donors compared to bladder cancer tissues by Western blotting. (D) Analysis of expression of MAN1B1 between paired erythrocytes and tumor tissues in bladder cancer patients by Western blotting. (E‐F) Representative flow cytometry plots of surface binding of anti‐human CD47 antibody clone B6H12 (anti‐hCD47) and SIRP‐α to red blood cells of healthy donors which were treated with DHT I (0.625‐10 µmol/L). (F) Values indicated MFI relative to control cells stained with the same reagent (n = 3 donors). P values in (F) were determined by a two‐tailed unpaired Student's t‐test, error bars represent mean ± SD. NS, not significant. Abbreviations: CD47, cluster of differentiation 47; SIRP‐α, signal‐regulatory protein alpha; MAN1B1, mannosidase alpha class 1B member 1; DHT I, dihydrotanshinone I; GAPDH, Glyceraldehyde‐3‐phosphate dehydrogenase; RBC, red blood cell; MFI, mean fluorescence intensity; BLCA, bladder urothelial carcinoma; SD, standard deviation.

FIGURE 6

ERK stabled MAN1B1 by regulating its interaction with E3 ligase HRD1 in bladder cancer. (A) Analysis of mRNA levels of MAN1B1 in bladder cancer by qRT‐PCR (n = 8). (B) Western blotting analysis of WCL derived from 5637 cells treated with trametinib (1 µmol/L) for 24 h before harvesting. (C) Western blotting analysis of WCL derived from J82 cells treated with trametinib (1 µmol/L) before harvesting. (D‐G) Western blotting analysis of WCL derived from bladder cancer cells transfected with KRAS‐G12D or ERK2 as indicated. (H) Analysis of mRNA level of MAN1B1 in 5637 cells which were stably transfected KRAS‐G12D by qRT‐PCR. (I) Analysis of interaction between ERK2 and MAN1B1 by IP. (J) Western blotting analysis of WCL derived from HEK293T cells co‐transfected MAN1B1‐FLAG with KRAS‐G12D or ERK2 as indicated. (K) Western blotting analysis of stability of MAN1B1 treated with 50 µg CHX when co‐transfected with or without ERK2. (L) Western blotting analysis of WCL and anti‐FLAG from lysates of sgControl or sgERK1/2 treated 293T using indicated antibodies. Cells were pretreated with 5 µmol/L MG132 for 12 h. (M‐N) Representative flow cytography (M) and quantification (N) depicted the process of macrophages mediated phagocytosis of J82 cells treated with trametinib In vitro compared to the control group. Boxes were representative of GFP⁺ CD11b⁺ macrophages. (O) Network view of predicted E3 ubiquitin ligase‐MAN1B1 interactions by UbiBrowser 2.0. (P) Analysis of interaction between HRD1 and MAN1B1 by IP. (Q) Western blotting analysis of HRD1 knockout in J82 cells. (R) Western blotting analysis of stability of MAN1B1 in J82 sgControl or sgMAN1B1 cells. (S‐T) Correlation between MAN1B1 and HRD1 in ten human fresh bladder cancer samples using Western blotting. N, matched normal tissue; T, tumor tissue. (U‐V) Analysis of MAN1B1‐HRD1 binding by IP when ERK was phosphorylated in J82 and 293T cells. (W) Analysis of MAN1B1‐HRD1 binding by IP when ERK was dephosphorylated in UMUC3 cells. P values (A, H and N) were determined by a two‐tailed unpaired Student's t‐test, error bars represent mean ± SD. P value (T) was calculated by the Pearson correlation test. NS, not significant; **P < 0.01. Abbreviations: CD47, cluster of differentiation 47; SIRP‐α, signal‐regulatory protein alpha; MAN1B1, mannosidase alpha class 1B member 1; WCL, whole cell lysates; IP, immunoprecipitation; HRD1, HMG‐CoA Reductase Degradation; ERK2, extracellular signal‐regulated kinase 2; KRAS‐G12D, Kirsten Rat Sarcoma Viral Oncogene Homolog G12D; GAPDH, Glyceraldehyde‐3‐phosphate dehydrogenase; EGF, epidermal growth factor; MG132, Carbobenzoxy‐L‐leucyl‐L‐leucyl‐L‐leucinal; GFP, green fluorescent protein; SYVN1, synoviolin 1; CD147, cluster of differentiation; DMSO, dimethyl sulfoxide; Vec, vector; CHX, cycloheximide; SD, standard deviation.