Androgen receptor affects the response to immune checkpoint therapy by suppressing PD-L1 in hepatocellular carcinoma - PubMed (original) (raw)

Androgen receptor affects the response to immune checkpoint therapy by suppressing PD-L1 in hepatocellular carcinoma

Guangyi Jiang et al. Aging (Albany NY). 2020.

Abstract

Hepatocellular carcinoma (HCC) is a heterogeneous malignancy with gender-related differences in onset and course. Androgen receptor (AR), a male hormone receptor, is critical in the initiation and progression of HCC. The role of AR in HCC has been mechanistically characterized and anti-AR therapies have been developed, showing limited efficacy. Immunotherapy targeting immune checkpoint proteins may substantially improve the clinical management of HCC. The mechanism by which AR influences HCC immune state remains unclear. In this study, we demonstrated that AR negatively regulated PD-L1, by acting as a transcriptional repressor of PD-L1. Notably, AR over-expression in HCC cells enhanced CD8+T function in vitro. We then verified the AR/PD-L1 correlation in patients. In animal experiment we found that lower AR expressed tumor achieved better response to PD-L1 inhibitor. Thus, AR suppressed PD-L1 expression, possibly contributing to gender disparity in HCC. Better understanding of the roles of AR during HCC initiation and progression will provide a novel angle to develop potential HCC immunotherapies.

Keywords: PD-1/PD-L1 pathway; androgen receptor; immune surveillance; tumor microenvironment.

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Conflict of interest statement

CONFLICTS OF INTEREST: The authors declare that they have no conflicts of interest.

Figures

Figure 1

Figure 1

Modulation of AR influence the expression of PD-L1 in HCC cells. (A–C) RT-qPCR analysis of three checkpoints in over-expression AR and down-expression AR HCC cells. (D) Western Blot of AR and PD-L1 in three HCC cell lines. (C) Flow cytometry of the membrane PD-L1. (E, F) Mean fluorescence intensity of membrane PD-L1. *P<0.05, ** P<0.01 and *** P<0.001.

Figure 2

Figure 2

Modulation of AR regulate the immune state. (A) Schematic diagram of PD1-PD-L1 binding assay. (B) The results of binding assay in three HCC cell lines. (C) Intracellular INF-r expression in CD8+T cells co-cultured with HCC cells. (D) Intracellular TNF-a expression in CD8+T cells co-cultured with HCC cells. (E) Secreted cytokine (granzyme B and perforin) in MHCC97H cells. (F) ELISA of serum secreted cytokine (granzyme B and perforin) in HCCLM3 cells. (G) T cell cytotoxicity assay in MHCC97H with different AR expression. (H) AR antagonist cause change of AR and PD-L1 in vitro. *P<0.05, ** P<0.01 and *** P<0.001.

Figure 3

Figure 3

AR activates PD-L1 transcription by binding to its promoter region. (A) Castration assay was performed in three HCC cell lines. (B) Predicted localization of AREs in PD-L1 promoter region (red). (C) Chromatin immunoprecipitation was performed in wild-type SK-Hep1 cells. The detecting primer was designed based on the prediction result of potential AREs. (D) Wild-type PD-L1 promoter construct was transfected into SK-Hep1 cells with internal control pRL-TK. Then, we performed luciferase reporter assays with manipulated AR to detect if AR could affect activation of PD-L1 promoter. (E) Luciferase reporter assays were performed after transfecting mutated 1st ARE into AR-overexpressed SK-Hep1 cells and AR knocked-down SK-Hep1 cells. *P<0.05, ** P<0.01 and *** P<0.001.

Figure 4

Figure 4

The negative correlation between AR and PD-L1 in vivo. (A) AR protein expression and membrane PD-L1 detection in patients’ samples (B) The correlation results between AR and PD-L1 (C) Representative images for scoring the AR IHC staining. (D) Representative images for scoring the PD-L1 IHC staining. (E) Representative images to show the comparison of AR and PD-L1 staining in the same patient. (F) Spearman correlation analysis for AR and PD-L1 based in our stained clinical samples (P value= 0.0039). (G) Survival curve analysis in different AR expression.

Figure 5

Figure 5

AR overexpression attenuated the effects of the PD-L1 inhibitor in vivo. (A) Establishment of overexpressed AR Hep1-6 and tested by western blot and flow cytometry. (B) Flow diagram of animal experiment. (C) The luminescence of tumor detected by IVIS system. (D) The growth curve of mice liver tumor represented by photon counts. (E) Picture of liver tumor and tumor infiltrating lymphocytes (TILs) detected by flow cytometry. (F) Flow diagram of animal experiment on castrated mice. (G) The luminescence of tumor detected by IVIS system. (H) The statistical results of the animal experiment. (I) Tumor infiltrating lymphocytes (TILs) detected by flow cytometry.

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