FUCA1 Reduces PD-L1 Fucosylation To Regulate Apoptosis And Migration In TNBC Cells

Objective: Breast cancer is a common malignancy that poses a serious threat to women’s health and life.Among them,Triple-Negative Breast Cancer is one of the most aggressive subtypes of breast cancer,which has a poor prognosis level compared with other breast cancer due to its lack of effective targeting treatment.PD-L1,as an immune checkpoint protein,has achieved good results in the treatment of lung cancer and melanoma,but it still has poor results in the treatment of TNBC.So,it is important to investigate its causes and use effective methods to intervene,and provide new ideas and strategies for immunotherapy of TNBC.It has been reported in the literature that the level of fucosylation is high in TNBC,and PD-L1 also has fucosylation modification.Whether modulating this fucosylation level in TNBC can enhance the therapeutic effect of PD-L1 in TNBC has not been reported in the literature.FUCA1 is a lysosomal enzyme that hydrolyzes the glycoprotein terminal core fucose.The purpose of this research was to use FUCA1 to modulate the level of PD-L1 fucosylation,and to investigate whether the alteration of PD-L1 fucosylation could affect the killing ability of TNBC cells on T cells,as well as the effect on apoptosis and migration of TNBC cells,and finally elucidate the mechanism.Our study provides theoretical support for new clinical treatment options for TNBC.Methods:(1)Analysis of FUCA1 expression as well as prognosis in clinical samples from the GEPIA database using bioinformatics methods.(2)To determine the difference of FUCA1 expression in serum and pathological tissues between TNBC and non-TNBC patients using Western Blot and Immunohistochemistry techniques.(3)The protein and gene expression of FUCA1 in MCF-7,MDA-MB-231 and BT-549 breast cancer cells were detected by Western Blot and Immunofluorescence and Real-Time q PCR,respectively.Finally,two TNBC cells,MDA-MB-231 and BT-549,were identified as the subjects for subsequent experimental studies.(4)Using CCK-8,Western Blot,and Immunofluorescence to determine the optimal concentration of ADR administration and to explore the effect of ADR on the regulation of FUCA1.(5)Overexpression of FUCA1,interfering FUCA1,interfering PD-L1 plasmids were constructed and transferred to MDA-MB-231 and BT-549 cells by transient transfection technique,and their ability to regulate PD-L1 fucosylation was determined by Western Blot,Lectin blot and Immunofluorescence assay.(6)Determine the effect of regulating the level of fucosylation of PD-L1 on apoptosis and migration of TNBC cells by co-culture with Jurkat T cells,Wound Healing Test,Flow Cytometry,and Western Blot.(7)The expression of molecules related to PI3K/AKT signaling pathway were detected by Western Blot,Immunofluorescence,and the alteration of PD-L1 glycosylation was detected by PI3 K pathway inhibitor(LY294002)to determine that FUCA1 reduces the fucosylation on the surface of PD-L1 is achieved through this signaling pathway.Results:(1)The results of GEPIA database analysis showed that patients with lower FUCA1 expression had poorer prognostic survival.(2)The expression levels of FUCA1 in serum and tissues of TNBC patients were significantly lower than the levels in nonTNBC patients.(3)The protein levels and gene levels of FUCA1 in TNBC cell lines MDA-MB-231 and BT-549 were lower than those in non-TNBC cell line MCF-7.(4)The appropriate concentration of ADR-induced FUCA1 expression was determined to be 1μM.(5)Treatment with different concentrations of ADR or to construct overexpressing FUCA1 could effectively regulate the expression of FUCA1 in TNBC cell lines,it also can simultaneously reduce the level of PD-L1 fucosylation.(6)After co-culture with Jurkat T cells,the reduced level of PD-L1 fucosylation promoted the killing of tumor cells by T cells,inhibited immune escape,and promoted apoptosis and inhibited migration of tumor cells.(7)FUCA1 reduces PD-L1 fucosylation levels by inhibiting the activation of PI3K/AKT signaling pathway.Conclusion: In this study,it was firstly clarified that FUCA1 expression was low in TNBC patients and this low expression was one of the reasons why PD-L1 showed a high fucosylation level.The use of ADR can increase the expression of FUCA1 while inhibiting PD-L1 deglycosylation level,which can affect the killing of TNBC cells by T cells as well as the apoptosis and migration of TNBC cells.This study provides a new theoretical basis for targeting PD-L1 fucosylation in TNBC treatment,and inhibition of core fucosylation is a unique strategy to reduce the binding of PD-1 to PD-L1 for the antiTNBC immune therapy in the future.