D-mannose facilitates immunotherapy and radiotherapy of triple-negative breast cancer via degradation of PD-L1

Abstract

Breast cancer is the most frequent malignancy in women worldwide, and triple-negative breast cancer (TNBC) patients have the worst prognosis and highest risk of recurrence. The therapeutic strategies for TNBC are limited. It is urgent to develop new methods to enhance the efficacy of TNBC treatment. Previous studies demonstrated that D-mannose, a hexose, can enhance chemotherapy in cancer and suppress the immunopathology of autoimmune diseases. Here, we show that D-mannose can significantly facilitate TNBC treatment via degradation of PD-L1. Specifically, D-mannose can activate AMP-activated protein kinase (AMPK) to phosphorylate PD-L1 at S195, which leads to abnormal glycosylation and proteasomal degradation of PD-L1. D-mannose-mediated PD-L1 degradation promotes T cell activation and T cell killing of tumor cells. The combination of D-mannose and PD-1 blockade therapy dramatically inhibits TNBC growth and extends the lifespan of tumor-bearing mice. Moreover, D-mannose-induced PD-L1 degradation also results in messenger RNA destabilization of DNA damage repair-related genes, thereby sensitizing breast cancer cells to ionizing radiation (IR) treatment and facilitating radiotherapy of TNBC in mice. Of note, the effective level of D-mannose can be easily achieved by oral administration in mice. Our study unveils a mechanism by which D-mannose targets PD-L1 for degradation and provides methods to facilitate immunotherapy and radiotherapy in TNBC. This function of D-mannose may be useful for clinical treatment of TNBC.

Keywords: D-mannose; PD-L1; immunotherapy; radiotherapy; triple-negative breast cancer.

Fig. 1.

D-mannose down-regulates PD-L1 in TNBC cells. (A and B) Western blot analysis of PD-L1 level in MDA-MB-231 cells (A) and BT-549 cells (B) treated with or without different hexoses as indicated. (C and D) qRT-PCR analysis of PD-L1 level in MDA-MB-231 cells (C) and BT-549 cells (D) treated with or without different hexoses as indicated. Values are means ± SD from n = 3 independent experiments. (E and F) Western blot analysis of PD-L1 level in MDA-MB-231 cells (E) and BT-549 cells (F) treated with 100 mM D-mannose for different time as indicated. (G and H) Western blot analysis of PD-L1 level in MDA-MB-231 cells (G) and BT-549 cells (H) treated with different concentrations of D-mannose as indicated for 72 h. (I and J) Flow cytometry analysis of membrane PD-L1 expression in MDA-MB-231 (I) and BT-549 cells (J) (stimulated with IFN-γ) under D-mannose treatment. Representative histograms and summarized mean fluorescent intensity (MFI) are shown. Values are means ± SD from n = 3 independent experiments. Statistical differences were determined by Student’s t test. ****P < 0.0001. (K) Western blot analysis of PD-L1 level in MDA-MB-231 cells treated with 25 mM D-mannose for different time under high glucose or glucose deprivation. (L) Western blot analysis of PD-L1 level in MDA-MB-231 cells treated with 25 mM D-mannose and decreasing concentrations of glucose as indicated for 72 h. (M) Western blot analysis of PD-L1 in MDA-MB-231 cells treated with 25 mM D-mannose and increasing concentrations of 2-DG as indicated for 72 h.

Fig. 2.

D-mannose disturbs PD-L1 glycosylation and stabilization through activating AMPK. (A and B) The half-life of PD-L1 under D-mannose treatment was determined by cycloheximide (CHX)-chase assay in 293T cells overexpressed with PD-L1 (A) and MDA-MB-231 cells (B). The quantifications are shown below. Values are means ± SD from n = 3 independent experiments. Statistical differences were determined by Student’s t test. *P < 0.05, **P < 0.01. (C) Western blot analysis of PD-L1 level in MDA-MB-231 cells treated with D-mannose in the absence or presence of proteasome inhibitor MG132, or lysosome inhibitor NH₄Cl, or autophagy inhibitor 3-MA. (D) Western blot analysis of PD-L1 ubiquitination level under D-mannose treatment in MDA-MB-231 cells stably expressing Flag-PD-L1. Ubiquitinated PD-L1 proteins were immunoprecipitated with Flag beads and blotted with HA antibody. (E) Western blot analysis of PD-L1 WT and NQ mutants in MDA-MB-231 cells treated with or without D-mannose. (F) Western blot analysis of PD-L1 as well as AMPK-α and p-AMPK-α in MDA-MB-231 cells treated with D-mannose for different time as indicated. (G) Western blot analysis of PD-L1 as well as AMPK-α and p-AMPK-α in MDA-MB-231 cells treated with different concentrations of D-mannose as indicated. (H) The effect of D-mannose on the interaction between PD-L1 and AMPK-α in MDA-MB-231 cells was determined by co-IP and Western blot. (I) The function of AMPK-α on D-mannose–induced PD-L1 degradation was determined by Western blot. AMPK-α WT and KO cells were treated with D-mannose (100 mM, 48 h). Levels of PD-L1 as well as AMPK-α and p-AMPK-α in MDA-MB-231 cells were examined by Western blot. (J) The effect of AMPK inhibition on D-mannose–induced PD-L1 degradation was determined by Western blot. MDA-MB-231 cells were pretreated with AMPK inhibitor dorsomorphin (10 μM, 6 h) before cells were treated with D-mannose (100 mM, 48 h). Levels of PD-L1 as well as AMPK-α and p-AMPK-α in MDA-MB-231 cells were examined by Western blot. (K) Western blot analysis of WT, S195A, S195D, and S195E mutants of PD-L1 in MDA-MB-231 cells. (L) Western blot analysis of WT, S195A, S195D, and S195E mutants of PD-L1 as well as AMPK-α and p-AMPK-α in MDA-MB-231 cells treated with or without D-mannose (100 mM, 48 h). (M) CHX-chase assay showing the degradation of PD-L1 WT and S195A mutant in MDA-MB-231 cells treated with D-mannose (100 mM, 48 h).

Fig. 3.

D-mannose promotes T cell activation and T cell killing of TNBC cells in vitro. (A) Immunostaining of PD-1 Fc chimera proteins on MDA-MB-231 cells treated with or without D-mannose (Scale bars, 100 μm). Quantitation of binding of PD-1/Fc were shown on the Right. Values are means ± SD from n = 3 independent experiments. Statistical differences were determined by Student’s t test. ***P < 0.001. (B) Western blot analysis of PD-1 levels in Jurkat T cells stimulated with or without PHA for 72 h. (C) qRT-PCR analysis of IL-2 expression in Jurkat T cells cocultured with control or D-mannose–pretreated MDA-MB-231 or BT-549 cells. Jurkat T cells were activated by PHA and then cocultured with MDA-MB-231 or BT-549 cells pretreated with or without D-mannose. Values are means ± SD from n = 3 independent experiments. Statistical differences were determined by Student’s t test. **P < 0.01, ***P < 0.001, ****P < 0.0001. (D) Western blot analysis of PD-L1 level in 4T1 mouse breast cancer cells treated with or without D-mannose. (E) qRT-PCR analysis of IL-2 expression in mouse primary CD8⁺ T cells cocultured with control or D-mannose–pretreated 4T1 mouse breast cancer cells. Mouse primary CD8⁺ T cells were preactivated with PMA and ionomycin and then cocultured with 4T1 mouse breast cancer cells pretreated with or without D-mannose. (F) qRT-PCR analysis of IFN-γ expression in mouse primary CD8⁺ T cells cocultured with control or D-mannose–pretreated 4T1 mouse breast cancer cells. Mouse primary CD8⁺ T cells were preactivated with PMA and ionomycin and then cocultured with 4T1 mouse breast cancer cells pretreated with or without D-mannose. Values are means ± SD from n = 3 independent experiments. Statistical differences were determined by Student’s t test. ****P < 0.0001. (G) Morphology of activated human T cells. (H) The effect of D-mannose on T cell killing of TNBC cells was determined. Control and D-mannose–treated MDA-MB-231 cells were cocultured with activated T cell for 48 h and then subjected to crystal violet staining. The quantification was shown on the Right. Data represent mean ± SD n = 3. ***P < 0.001.

Fig. 4.

D-mannose synergizes with PD-1 antibody to inhibit TNBC growth. (A) Schematic representation of the animal experiment process. (B) Representative tumors of 4T1 cells in BALB/c mice treated with D-mannose or/and anti–PD-1 antibody. (C) Tumor growth of 4T1 cells in BALB/c mice following treatment with D-mannose or/and anti–PD-1 antibody was determined. Tumors were measured at the indicated time points. n = 8 mice per group. Statistical differences were determined by ordinary one-way ANOVA. ns, no significance, ****P < 0.0001. (D) Kaplan–Meier survival curves for mice injected with 4T1 cells and treated with D-mannose or/and anti–PD-1 antibody. The P value, comparing every two groups, was determined using log-rank test and is shown in the table Below the figure. (E) Western blot analysis of PD-L1 level in 4T1 tumor tissues as indicated. (F) Immunohistochemistry showing CD8⁺ T cell infiltration and granzyme B expression in the 4T1 tumor tissues as indicated (Scale bars, 100 μm). (G) Quantifications of images in (F). Data represent mean ± SD from six independent samples of each group. Statistical differences were determined by ordinary one-way ANOVA. ns, no significance, *P < 0.05, **P < 0.01, ***P < 0.001, ****P < 0.0001.

Fig. 5.

D-mannose inhibits PD-L1–mediated DDR and sensitizes TNBC cells to IR. (A and B) qRT-PCR analysis of BRCA1, RAD50, and MRE11 mRNA levels under D-mannose treatment in MDA-MB-231 cells (A) and BT-549 cells (B). Values are means ± SD from n = 3 independent experiments. Statistical differences were determined by Student’s t test. **P < 0.01, ***P < 0.001. (C and D) Western blot analysis of BRCA1, RAD50, and MRE11 levels in MDA-MB-231 cells (C) and BT-549 cells (D) treated with D-mannose. (E) qRT-PCR analysis of BRCA1, RAD50, and MRE11 mRNA levels in control and D-mannose–treated MDA-MB-231 cells after treating with the transcription inhibitor actinomycin D for different time as indicated. Values are means ± SD from n = 3 independent experiments. Statistical differences were determined by Student’s t test. *P < 0.05, ***P < 0.001, ****P < 0.0001. (F) Western blot analysis of PD-L1 in control and PD-L1 KO MDA-MB-231 cells. (G) qRT-PCR analysis of BRCA1, RAD50, and MRE11 mRNA levels in control or PD-L1 KO MDA-MB-231 cells following D-mannose treatment. Values are means ± SD from n = 3 independent experiments. Statistical differences were determined by Student’s t test. ns, no significance, **P < 0.01, ***P < 0.001. (H) Western blot analysis of BRCA1, RAD50, and MRE11 levels in control or PD-L1 KO MDA-MB-231 cells under D-mannose treatment. (I) Immunostaining of γ-H2AX in MDA-MB-231 cells with IR (3Gy) and D-mannose treatment as indicated. (J) Quantifications of images in (I). Data represent mean ± SD from three independent samples of each group. Statistical differences were determined by Student’s t test. **P < 0.01. (K) The growth of control and D-mannose–treated MDA-MB-231 cells under IR (3Gy) treatment was determined by colony formation assay. (L) Quantifications of images in (K). Data represent mean ± SD. Statistical differences were determined by Student’s t test. ***P < 0.001.

Fig. 6.

D-mannose sensitizes TNBC to radiotherapy. (A) Schematic representation of the animal experiment process. (B) Tumor growth of 4T1 cells in BALB/c mice treated with D-mannose or/and IR was determined. n = 10 mice per group. Statistical differences were determined by ordinary one-way ANOVA. ns, no significance, ****P < 0.0001. (C) Representative tumors resected from each group of mice that received different treatment as indicated. (D) The weight of tumors resected from each group of mice that received different treatment as indicated was analyzed. Data represent mean ± SD, n = 10 mice per group. Statistical differences were determined by ordinary one-way ANOVA. ns, no significance, ***P < 0.001, ****P < 0.0001. (E) Western blot analysis of PD-L1 level in 4T1 tumor tissues as indicated. (F) Left: Immunohistochemistry showing γ-H2AX expression in the 4T1 tumor tissues (Scale bars, 100 μm). Right: Quantifications of the immunohistochemistry images. Data represent mean ± SD from 15 independent samples of each group. Statistical differences were determined by ordinary one-way ANOVA. **P < 0.01, ***P < 0.001, ****P < 0.0001.

Fig. 7.

D-mannose induces PD-L1 abnormal glycosylation and degradation through proteasome, promoting cancer immunotherapy and radiotherapy. Membrane localized PD-L1 can interact with PD-1 on T cells to inhibit T cell activation. While intracellular PD-L1 can serve as an RNA-binding protein and stabilize the mRNAs of BRCA1, RAD50, and MRE11 to promote the DDR in tumor cells, which makes tumor cells resistant to IR treatment. Under D-mannose treatment, PD-L1 is phosphorylated by AMPK at S195, leading to its abnormal glycosylation and proteosomal degradation, which synergizes with anti–PD-1 antibody to promote T cell activation and facilitates mRNA decay of BRCA1, RAD50, and MRE11, thereby sensitizing tumor cells to immunotherapy and radiotherapy.