PD-L1 is overexpressed on breast cancer stem cells through notch3/mTOR axis

Abstract

ABSTRACT The T-cell inhibitory molecule PD-L1 is expressed on a fraction of breast cancer cells. The distribution of PD-L1 on the different subpopulations of breast cancer cells is not well-defined. Our aim was to study the expression level of PD-L1 on breast cancer stem-like (CSC-like) cells and their differentiated-like counterparts. We used multi-parametric flow cytometry to measure PD-L1 expression in different subpopulations of breast cancer cells. Pathway inhibitors, quantitative immunofluorescence, cell sorting, and western blot were used to investigate the underlying mechanism of PD-L1 upregulation in CSC-like cells. Specifically, PD-L1 was overexpressed up to three folds on breast CSC-like cells compared with more differentiated-like cancer cells. Functional in vitro and in vivo assays show higher stemness of PD-L1hi as compared with PD-L1lo cells. Among different pathways examined, PD-L1 expression on CSCs was partly dependant on Notch, and/or PI3K/AKT pathway activation. The effect of Notch inhibitors on PD-L1 overexpression in CSCs was completely abrogated upon mTOR knockdown. Specific knockdown of different Notch receptors shows Notch3 as a mediator for PD-L1 overexpression on CSCs and important for maintaining their stemness. Indeed, Notch3 was found to be overexpressed on PD-L1hi cells and specific knockdown of Notch3 abolished the effect of notch inhibitors and ligands on PD-L1 expression as well as mTOR activation. Our data demonstrated that overexpression of PD-L1 on CSCs is partly mediated by the notch pathway through Notch3/mTOR axis. We propose that these findings will help in a better design of anti-PD-L1 combination therapies to treat breast cancer effectively.