PD-L1 promotes OCT4 and Nanog expression in breast cancer stem cells by sustaining PI3K/AKT pathway activation

Abstract

The expression of PD-L1 in breast cancer is associated with estrogen receptor negativity, chemoresistance and epithelial-to-mesenchymal transition (EMT), all of which are common features of a highly tumorigenic subpopulation of cancer cells termed cancer stem cells (CSCs). Hitherto, the expression and intrinsic role of PD-L1 in the dynamics of breast CSCs has not been investigated. To address this issue, we used transcriptomic datasets, proteomics and several in vitro and in vivo assays. Expression profiling of a large breast cancer dataset (530 patients) showed statistically significant correlation (p < 0.0001, r = 0.36) between PD-L1 expression and stemness score of breast cancer. Specific knockdown of PD-L1 using ShRNA revealed its critical role in the expression of the embryonic stem cell transcriptional factors: OCT-4A, Nanog and the stemness factor, BMI1. Conversely, these factors could be induced upon PD-L1 ectopic expression in cells that are normally PD-L1 negative. Global proteomic analysis hinted for the central role of AKT in the biology of PD-L1 expressing cells. Indeed, PD-L1 positive effect on OCT-4A and Nanog was dependent on AKT activation. Most importantly, downregulation of PD-L1 compromised the self-renewal capability of breast CSCs in vitro and in vivo as shown by tumorsphere formation assay and extreme limiting dilution assay, respectively. This study demonstrates a novel role for PD-L1 in sustaining stemness of breast cancer cells and identifies the subpopulation and its associated molecular pathways that would be targeted upon anti-PD-L1 therapy.

Keywords: AKT; Nanog; OCT4A; PD-L1; breast cancer; cancer stem cells; stemness.

Figure 1

PD‐L1 expression in breast cancer is significantly associated with stemness score. Scatter plot of PD‐L1 mRNA expression level against the stem‐like score, which was calculated based on the expression of 100 stem‐cell‐associated genes as described in methods, in the TCGA breast cancer gene expression dataset (n = 530). Pearson correlation coefficients (r) and associated p values (p) for the correlation test is shown. [Color figure can be viewed at wileyonlinelibrary.com]

Figure 2

PD‐L1 expression is important for the expression of stemness factors. The expression level of OCT‐4A, Nanog and BMI‐1 is shown in PD‐L1‐knockdown clones (a & b) of MDA‐MB‐231 cells compared with the control cells (Sh‐Cont). (a) Bar graph showing the expression level (measured as mean fluorescence intensity, MFI) of nuclear OCT‐4A, Nanog and BMI1 (top) and the percentage of cells expressing them (bottom). Data is normalized on the control (Sh‐Cont) and displayed as mean of 4 independent experiments ± SEM as measured by immunofluorescence and quantified using BD pathway 855 system. (b) Representative immunofluorescent images of the PD‐L1‐knockdown clones and the control cells (at 200× magnification) from one of the 4 experiments. (c) Representative western blot images showing downregulation of nuclear OCT‐4A, Nanog and BMI1 in PD‐L1 knockdown cells. * indicates statistical significance (p < 0.05).

Figure 3

PD‐L1 knockdown impaired the phosphorylation of AKT. (a) Bar graph showing the expression level (measured as MFI) of phospho (S473)‐AKT or phospho (S235/236)‐S6 in PD‐L1‐knockdown clones (a & b) of MDA‐MB‐231 cells compared with the control cells (Sh‐Cont). Data are normalized to the control (Sh‐Cont) nuclear MFI of phospho AKT (left) or the control cytoplasmic phospho S6 (right) and displayed as mean of 4 independent experiments ± SEM as measured by immunofluorescence and quantified using BD pathway 855 system. (b) Representative immunofluorescent images of the PD‐L1‐knockdown clones and the control cells (at 200× magnification) from one of the experiments. (c) Western blot showing phospho AKT expression following PD‐L1 knockdown in MDA‐MB‐231 cells. (d) Bar graph showing the expression level of phospho‐AKT, OCT‐4A and Nanog in PD‐L1 knockdown clones and control ± PI3K/AKT/mTOR inhibitors as compared with untreated cells as mean of 10 independent experiments ± SEM. (e) Representative immunofluorescent images showing staining of phospho (S473)‐AKT, OCT‐4A, Nanog (at 200× magnification) from one of the 10 experiments in D for clone Sh‐PD‐L1 (b). * indicates statistical significance (p < 0.05).

Figure 4

PD‐L1‐mediated PI3K/AKT/mTOR activity promotes phosphorylation of OCT‐4 (T235). (a) Bar graph showing the expression level of phospho (T235) OCT‐4 in PD‐L1‐knockdown clones (a & b) of MDA‐MB‐231 cells compared with the control cells (Sh‐Cont). Data are normalized on the nuclear MFI of the control (Sh‐Cont) and displayed as mean of 3 independent experiments ± SEM as measured by immunofluorescence and quantified using BD pathway 855 system. (b) Representative immunofluorescent images of the PD‐L1‐knockdown clones (a & b) and the control cells (at 200× magnification) from one of the experiments. (c) Western blot showing expression of phospho OCT4 following PD‐L1 knockdown in MDA‐MB‐231 cells in the presence or absence of PI3K/AKT/mTOR inhibitors. (d) Schematic diagram showing the effect of PD‐L1 on stemness of breast cancer cells via a PI3K/AKT‐dependent and independent pathways. Solid blue lines indicate a demonstrated direct effect (thick = strong, thin = mild). Dashed lines indicate a demonstrated effect (direct or indirect). Solid red line indicates a demonstrated effect by other studies. * indicates statistical significance (p < 0.05).

Figure 5

(a) PD‐L1 is overexpressed by CSCs and is important for its self‐renewal of potential. (a) PD‐L1 knockdown abrogates the self‐renewal of CSCs in the tumorsphere assay. Bar graph showing the number of tumorspheres formed from 1000 cells (mean± SD, n = 3) for three consecutive passages. (b) Representative immunofluorescent images showing PD‐L1 expression in sorted CSCs (Ep‐CAM+/CD44^high/CD24^low). (c) Bar graph showing percentage of CSCs (Ep‐CAM+/CD44^high/CD24^low) in PD‐L1 knockdown cells compared with the control as measured by flow cytometry (mean± SEM, n = 6). * indicates statistical significance (p < 0.05).