The Inhibition of PD-L1 Glycosylation Abolishes Immune Suppression Activity and Induces Breast Cancer Tumor Regression
Introduction. Breast cancer is the second most common cancer found in women worldwide. Triple negative breast cancer (TNBC) is a subtype that lacks estrogen and progesterone receptors and HER2 proteins, but expresses higher PD-L1 levels1,2. Patients with TNBC have a worse prognosis compared to patients with other subtypes3. Therefore, some studies have focused on the immune checkpoint pathways involved in tumor growth, such as the PD-1/PD-L1 pathway. Activation of this pathway contributes to T-cell anergy and tumor growth4. The present study revealed that glycosylation of PD-L1 is crucial for PD-L1 and PD-1 binding and could serve as a potential therapy for TNBC3. Methods. Protein PD-L1 was treated with PNGaseF, a glycosidase, and a Western blot analysis was performed to examine the migration patterns of glycosylated versus non-glycosylated PD-L1. An In vitro receptor ligand binding assay determined the importance of glycosylation in ligand binding by knockdown and re-expression of either gPD-L1 or ngPD-L1. A T-cell killing assay with immunofluorescence was used to measure T-cell activities with gPD-L1 and ngPD-L1. Mice were inoculated in vivo with 4T1 tumor cells to express gPD-L1 or ngPD-L1, and the 4T1 cell levels were then measured to determine tumor growth in both groups. For potential clinical applications, antibodies against PD-L1 were utilized. Antibodies, STM004 and STM108, were administered to mice to observe PD-1 binding and tumor growth, and PD-L1 internalization was observed by fluorescence tagged STM108 and lysosomes3. Results. In the Western blot, ngPDL1 traveled further, due to lower molecular weight, confirming the presence of a glycan structure and its potential as a therapeutic target site. The receptor ligand binding assay showed less PD-1 binding to ngPD-L1 compared to gPD-L1, indicating that blocking PD-L1 glycosylation results in decreased pathway activation3. Furthermore, the T-cell killing assay demonstrated higher T-cell activities for ngPD-L1 compared to gPD-L1 and blocking PD-L1 led to T-cell reactivation3,5. Tumor regression was also seen in mice expressing ngPD-L1 and mice with PD-L1 antibodies1,3. Additionally, STM108 showed PD-L1 internalization and degradation by lysosomes. Conclusions. PD-L1 glycosylation was essential for the activation of the PD-1/PD-L1 pathway, which led to tumor growth and T-cell anergy3. By blocking PD-L1, the T-cell population may be reactivated, and tumor growth diminished3,5. PD-L1 antibodies have shown to reduce PD-1 binding and tumor growth, and STM108 showed PD-L1 internalization, which decreased the likelihood of treatment toxicity1,3. These data suggest that PD-L1 antibodies could be a promising treatment for breast cancer patients3,6.
- Emens LA, Cruz C, Eder JP, et al. Long-term Clinical Outcomes and Biomarker Analyses of Atezolizumab Therapy for Patients With Metastatic Triple-Negative Breast Cancer: A Phase 1 Study. JAMA Oncol. 2019;5(1):74-82. doi:10.1001/jamaoncol.2018.4224
- Qin G, Wang X, Ye S, et al. NPM1 upregulates the transcription of PD-L1 and suppresses T cell activity in triple-negative breast cancer. Nat Commun. 2020;11(1):1669. Published 2020 Apr 3. doi:10.1038/s41467-020-15364-z
- Li CW, Lim SO, Chung EM, et al. Eradication of Triple-Negative Breast Cancer Cells by Targeting Glycosylated PD-L1. Cancer Cell. 2018;33(2):187-201.e10. doi:10.1016/j.ccell.2018.01.009
- Schütz F, Stefanovic S, Mayer L, von Au A, Domschke C, Sohn C. PD-1/PD-L1 Pathway in Breast Cancer. Oncol Res Treat. 2017;40(5):294-297. doi:10.1159/000464353
- Jiao S, Xia W, Yamaguchi H, et al. PARP Inhibitor Upregulates PD-L1 Expression and Enhances Cancer-Associated Immunosuppression. Clin Cancer Res. 2017;23(14):3711-3720. doi:10.1158/1078-0432.CCR-16-3215
- Tang T, Huang X, Zhang G, Hong Z, Bai X, Liang T. Advantages of targeting the tumor immune microenvironment over blocking immune checkpoint in cancer immunotherapy. Signal Transduct Target Ther. 2021;6(1):72. Published 2021 Feb 20. doi:10.1038/s41392-020-00449-4