ALG3 predicts poor prognosis and increases resistance to anti-PD-1 therapy through modulating PD-L1 N-link glycosylation in TNBC

Abstract

Objective: The aim of this study was to assess the prognostic significance of α-1,3-mannitrotransferase (ALG3) in triple-negative breast cancer (TNBC) and investigate its impact and potential mechanism on the efficacy of anti-PD-1 therapy.

Methods: Bioinformatics analysis was used to examine the expression of ALG3 in cancer patients using UACLAN and other databases. The associations of the ALG3 gene and the clinicopathological features of breast cancer were examined with bc-GenExMiner database. Correlation between ALG3 expression and survival was further established utilizing the Kaplan-Meier Plotter database. Immunohistochemistry (IHC) was used to analyze the expression of ALG3 in cohort of breast cancer patients from Hubei cancer hospital to confirmed the prognostic value of ALG3 in TNBC. The effect of ALG3 on the levels of infiltrating immune cells was also analyzed. And the mutation module within cBioPortal was utilized to visualize ALG3 mutations in BRCA. The CRISPR/Cas9 technique was used to establish ALG3 low-expression TNBC cell lines. Influence of ALG3 expression on cancer cell proliferation and chemotherapeutic responsiveness was scrutinized in vitro. Animal models were constructed to evaluate the alteration of tumor sensitivity to anti-PD-1 therapy with decreased ALG3 expression. And flow cytometry and IHC were used to investigate the tumor immune microenvironment. Association of PD-L1 Glycosylation and ALG3 expression were also investigated by western blot.

Results: ALG3 expression was elevated in TNBC and was strikingly linked to unfavorable clinical features such as lymphatic node metastasis, high NPI, advanced stage and age, etc. Furthermore, high ALG3 expression was associated with shorter OS in TNBC patients. Mechanistically, ALG3 expression was negatively correlated with the infiltration of CD8⁺ T cells, CD4⁺ T cells, and NK cells. ALG3-KO cells had increased sensitivity to chemotherapeutic agents. In animal models, the volume of ALG3-KO tumors was lower than the control group with immunotherapy. ALG3-KO tumors showed an increased proportion of CD8⁺ T cells, while a decreased proportion of regulatory T cells and M2-type macrophages. The expression level of PD-L1 protein was not affected by ALG3 level, but the glycosylation level was significantly decreased in tumor. Similarly, the glycosylation level of PD-L1 is reduced in ALG3-KO cell in vitro. Additionally, ALG3 knockout lead to reduced tolerance of tumor cells to IFN-γ, thereby enhancing the efficacy of immunotherapy.

Conclusion: ALG3 is a potential biomarker for poor prognosis of TNBC and may reduce the efficacy of immunotherapy by modulating the tumor microenvironment and glycosylation of PD-L1.

Keywords: ALG3; Immunotherapy; PD-L1; Prognosis; Triple negative breast cancer.