HIF1α-dependent and independent pathways regulate the expression of PD-L1 in prostate cancer

Abstract

PD-L1/PD-1 pathway is a major pathway exploited by human cancer types, which is a target for current immunotherapy. We investigated tumor microenvironmental factors involved in PD-L1 induction in prostate cancer (PC). We studied the expression of PD-L1 in a series of 66 PCs, in parallel with the expression of hypoxia- and acidity-related immunohistochemical markers (Hypoxia-inducible factor HIF1α, and lactate dehydrogenase LDHA) and tumor-infiltrating lymphocyte TIL density. Experiments with three PC cell lines, the 22Rv1, DU145, and PC3 were conducted focusing on the inducibility of PD-L1 by hypoxia, acidity, lymphocyte interactions, and radiation. In tissues, PD-L1 expression by cancer cells was directly related to PD-L1 expression by TILs and macrophages (p < 0.05), and the overexpression of HIF1α and LDH5 (p < 0.05). TIL density was inversely related to ΗΙF1α (p = 0.02). Exposure of PC cell lines to hypoxia strongly induced PD-L1 and protein and mRNA levels, directly controlled by HIF1α function (p < 0.001). Irradiation with 20 Gy had no apparent effect on PD-L1 expression. Culturing PC cell lines with culture medium (CM) from PBMCs strongly induced PD-L1 at protein and mRNA levels, independently from HIF1α, which was also confirmed when cells were incubated with Interferon-γ (p < 0.001). It is concluded that the combination of anti-PD-L1/PD-1 immunotherapy with hypoxia/HIF-targeting may be important in the treatment of specific subgroups of PC patients.

Keywords: HIF1α; Hypoxia; IFNγ; PD-L1; Prostate cancer; Radiotherapy.

Fig. 1

Immunohistochemical analysis of PC tissues: A Typical membrane expression of PD-L1 in cancer cells (arrows; magnification × 40); B Expression of PD-L1 in stroma infiltrating lymphocytes and monocytes (arrows; magnification × 40); C A PC tumor with nuclear and cytoplasmic expression of HIF1α (arrows; magnification × 20); D A PC tumor with nuclear and cytoplasmic expression of LDH5 (arrows; magnification × 20); E The % of cancer cells expressing HIF1α and LDH5 according to the expression of PD-L1 (negative vs. positive). Box and whiskers graphs showing the median value, range, and 25th/75th percentiles. (F,G,H,I). Basal levels of PD-L1 expression: F western blot images showing the basal expression levels of PD-L1 protein in PC cell lines 22Rv1, DU145, and PC3 in parallel with two human fibroblast cell lines (HFL1 and MRC5); G band densitometry of the western blot (p values are referred to the comparison between 22Rv1 and the other two PC cell lines); H typical immunocytochemical expression of PD-L1 expression levels in PC cell lines 22Rv1, DU145, PC3 (magnification × 40); I RT-PCR analysis of the mRNA expression of the PD-L1 gene, in PC cell lines DU145, PC3, in comparison with the expression in 22Rv1. (***p < 0.001). (Abbreviations: M = marker, *** = p < 0.001)

Fig. 2

PD-L1 expression under hypoxic, acidic conditions and exposure to the HIF1α-inhibitor chrysin: A & B Western blot images and densitometry analysis of the expression of protein PD-L1. C RT-PCR analysis showing the PD-L1 mRNAs changes in the 22Rv1, DU145, and PC3 PC cell lines exposed to hypoxia and acidity. D RT-PCR analysis of the mRNA expression of the HIF1α gene under normoxic conditions of the 22Rv1, DU145, PC3 shHIF1α cell lines in comparison with the control cell lines and cell lines transfected with a non-coding sequence. E PD-L1 expression in western blot analysis of control PC cell lines, cell lines transfected with a non-coding sequence, and PC cell lines with permanent silencing of HI1α gene, and F band densitometry after exposure to hypoxia. G Western blot images of the effect of 50 μmol/L of chrysin on HIF1α protein levels in DU145 and PC3 prostate cancer cell lines and band densitometry. H Western blot images of the effect of 50 μmol/L of Chrysin on PD-L1 protein levels in DU145 and PC3 prostate cancer cell lines and band densitometry (*p < 0.05, **p < 0.01, ***p < 0.001). (Abbreviations: M = marker, NC = non-coding, shHIF1α = silenced HIF1α with small hairpin RNA, NS = not significant, * = p < 0.05, ** = p < 0.01, *** = p < 0.001)

Fig. 3

PD-L1 expression and response to direct 20 Gy cancer cell irradiation, exposure to culture medium from 20 Gy irradiated cancer cells (ICM; 2-day culture after irradiation) and culture medium from untreated PBMCs (PCM, 2-day culture): Western blot images A and band densitometry B showing the expression pattern of PD-L1 of the 22Rv1, DU145, PC3 (control, transfected with non-coding NC sequence or with shHIF1α) cell lines (*p < 0.05, **p < 0.01, ***p < 0.001).). (Abbreviations: M = marker, NC = non-coding, shHIF1α = silenced HIF1α with small hairpin RNA, ICM = culture medium form irradiated cancer cells, PCM = culture medium from untreated PBMCs, * = p < 0.05, ** = p < 0.01, *** = p < 0.001)

Fig. 4

Effect of IFNγ on PD-L1 expression: A western blot images and B band densitometry showing the expression pattern of PD-L1 protein in control, nc- and sh- 22Rv1, DU145, PC3 cell lines after exposure to 25 ng/ml of IFNγ for 48 h. C RT-PCR analysis of the mRNA expression of the PD-L1 gene (*p < 0.05, **p < 0.01, ***p < 0.001). (Abbreviations: M = marker, NC = non-coding, shHIF1α = silenced HIF1α with small hairpin RNA, IFNγ = interferon gamma, * = p < 0.05, ** = p < 0.01, *** = p < 0.001)

Fig. 5

Two distinct cellular pathways regulating PD-L1 up-regulation. Pathway 1: HIF1α overexpression, whether hypoxia induced or inherently up-regulated, triggers PD-L1 overexpression on cancer cell membranes, producing a PD-L1-rich tumor (red structure), characterized by a hypoxic and/or acidic TME (gray). Anti-PD-1/PD-L1 immunotherapy neutralizes the anti-immune PD-L1 enrichment (light blue). However, due to hypoxic/acidic conditions, cytotoxic T-cells (yellow/green structures) cannot proliferate and act in this adverse TME. Blockage of HIF1α activity will have a dual effect by repressing PD-L1 expression and reducing anaerobic metabolism and acidity. This may assist cytotoxic T-cells in thriving in the tumor and exerting antitumor cytotoxicity. Pathway 2: Constitutive or Radiotherapy-induced IFNγ produced by TILMs up-regulates PD-L1 expression by cancer cells, blocking cytotoxic T-cell activity. Anti-PD-1/PD-L1 immunotherapy blocks PD-L1 and facilitates cytotoxic T-cell activity, provided that this pathway does not co-exist with the hypoxia-driven one