Regulation of PD-L1 expression in the tumor microenvironment

Programmed death-ligand 1 (PD-L1) on cancer cells engages with programmed cell death-1 (PD-1) on immune cells, contributing to cancer immune escape. For multiple cancer types, the PD-1/PD-L1 axis is the major speed-limiting step of the anti-cancer immune response. In this context, blocking PD-1/PD-L1 could restore T cells from exhausted status and eradicate cancer cells. However, only a subset of PD-L1 positive patients benefits from α-PD-1/PD-L1 therapies. Actually, PD-L1 expression is regulated by various factors, leading to the diverse significances of PD-L1 positivity. Understanding the mechanisms of PD-L1 regulation is helpful to select patients and enhance the treatment effect. In this review, we focused on PD-L1 regulators at the levels of transcription, post-transcription, post-translation. Besides, we discussed the potential applications of these laboratory findings in the clinic.


Background
In physiological conditions, the activities of T cells are intricately regulated. T cell immunity selectively eliminates pathogens and abnormal cells but avoids attacking normal cells, termed immune homeostasis [1]. Programmed cell death-1 (PD-1, which is encoded by PDCD1) and programmed death-ligand 1 (PD-L1, which is encoded by CD274) are vital proteins in maintaining immune homeostasis [2]. The PD-1/PD-L1 pathway restrains the hyperactivation of immune cells and prevents autoimmune diseases [3]. However, in the tumor microenvironment (TME), the PD-1/PD-L1 axis is hijacked by cancer cells to escape immune surveillance [4]. The overexpressed PD-L1 on cancer cells binds to the PD-1 on tumor-infiltrating lymphocytes (TILs), which counteracts the TCR-signaling cascade by phosphorylating SHP-2 [5,6]. As a result, T cell activation is impaired.
Apart from cancer cells, some other types of cells in the TME, such as macrophages, dendritic cells (DCs), activated T cells, as well as cancer-associated fibroblasts, also express PD-L1 [7]. These components orchestrate an immunosuppressive microenvironment, supporting tumor growth.
On the contrary, patients with PD-L1 negative tumors might respond to α-PD-1/PD-L1 treatment when undergoing combination therapies that promote T cell infiltration [22]. Therefore, an in-depth understanding of PD-L1 regulation is valuable for efficacy prediction and patient selection. In this review, we summarized the latest advances of PD-L1 regulation, including genomic alterations, epigenetic modification, transcriptional regulation, post-transcriptional modification, and post-translational modification. Moreover, we discussed the potential applications of these findings in the clinic.

Inflammatory Signaling Interferon (IFN) and IL-6
As a negative feedback for inflammation, PD-L1 could be upregulated by multiple inflammatory signaling pathways to restrain T cells' hyperactivity ( Fig. 1). Generally believed, IFN-γ is the prominent stimulator contributing to the inducible PD-L1 expression [43].
During cancer progression, the IFN-γ-derived PD-L1 promotes cancer immune escape [3]. In the TME, activated T cells and NK cells generate most IFN-γ. Then, IFN-γ binds to type II interferon receptor, activating the JAK-STAT signaling (mainly through STAT1) [44,45]. Subsequently, the expression of several transcriptional factors is upregulated, especially interferon-responsive factors (IRFs). IRF-1 is the vital downstream component of STAT1 upon IFN-γ treatment [46,47]. In hepatocellular carcinoma, it was identified that two elements (IRE1/2) in the 5′-flanking region of the CD274 promoter were the binding sites of IRF-1, which participated in regulating PD-L1 transcription [48]. Notably, the intactness of JAK-STAT-IRF1 pathway is also related to the response to α-PD-1/PD-L1 therapy. The effect of α-PD-1/ PD-L1 treatment is limited in tumors with mutations in JAK1 and JAK2 [49]. It was speculated that these tumors might not rely on the PD-1/PD-L1 pathway to escape immune surveillance [49].
The effect of TGF-β on PD-L1 regulation is still unclear. Although some previous studies indicated that TGF-β downregulated PD-L1 expression in renal tubular epithelial cells and monocytes [58,59], TGF-β mainly had a positive impact on the PD-L1 expression in the TME. In NSCLC cells, exogenous TGF-β increased the CD274 transcription probably by Smad-binding elements [60]. The expression of phosphorylated-Smad2 was significantly increased in PD-L1 positive NSCLC patients [60]. Apart from cancer cells, TGF-β could increase PD-L1 expression on DCs in the TME [61].

Oncogenic Signaling
Besides inflammatory stimuli, growing evidence suggests that hyperactive oncogenic pathways play a vital role in PD-L1 expression (Fig. 1). Therefore, α-PD-1/PD-L1 therapies might have a synergistic effect with oncogenic signal-targeting treatments.

Epidermal Growth Factor Receptor (EGFR)
In lung epithelial cells, the mutated EGFR pathway (EGFR T790M) increased PD-L1 expression [62]. For lung cancer cells, PD-L1 expression was impaired after EGFR tyrosine kinase inhibitor (TKI) treatment [62]. In murine EGFR-driving lung cancer models, α-PD-1 effectively reversed T cell exhaustion and retarded tumor growth [62]. The results indicated that the mutant EGFR pathway facilitated tumor to escape from immune surveillance [62]. However, a clinical study showed that EGFR-mutant NSCLC patients tended to resist α-PD-1 therapy [63]. The authors found that although some EGFR-mutant NSCLCs were PD-L1 positive, the concurrent PD-L1 upregulation and abundant TILs were rare [63]. The lack of a pre-existing inflammatory TME might limit the effect of α-PD-1/PD-L1 treatment [63]. The low response rate in EGFR-mutant patients was reported by other investigators [64,65].

Mitogen-activated protein kinase (MAPK)
MAPK is a well-studied oncogenic pathway, which counts for nearly 40% of human cancer cases [66]. According to TCGA database, the CD274 mRNA level was significantly positively related to RAS-or MEK-activation scores in NSCLC patients [67]. In lung adenocarcinoma cells, activating EGF-MAPK signaling increased the mRNA and protein levels of PD-L1 [67]. Inhibiting MAPK signaling by MEK inhibitor (Selumetinib) counteracted the EGF-and IFN-γ-stimulated upregulation of CD274 mRNA and PD-L1 protein [67]. In melanoma cells, the activated NRAS-RAF-MEK1/2-ERK-c-Jun axis enhanced the transcription of CD274 [68]. Moreover, in pancreatic cancer, myeloid cells induced PD-L1 expression on tumor cells by activating EGFR-MAPK pathway [69]. After MEK inhibitor treatment, the levels of p-ERK and PD-L1 were decreased, and this reduced PD-L1 led to a higher sensitivity to α-PD-1 treatment in murine pancreatic tumors [69].

Myc
As a transcription factor regulating cell differentiation, proliferation, and apoptosis, Myc is overexpressed in various cancers [95]. Knocking down or inhibiting Myc in cancer cells reduced CD274 mRNA and PD-L1 protein [96][97][98][99]. The results of the ChIP-seq assay showed that Myc could bind to the CD274 promoter [96]. However, in some particular types of cancer, Myc negatively regulated PD-L1 expression. In hepatocellular carcinoma cells, inhibiting Myc increased the IFN-γ-stimulated PD-L1 expression [100]. Besides, in the murine MycCaP tumor model, Myc inhibitor treatment promoted T cell infiltration, enhanced antitumor immune response, but simultaneously upregulated PD-L1 expression [101]. This PD-L1 upregulation was induced by immune response [101].

Met
Alterations in the Met gene were reported in multiple types of cancers [105,106]. In primary lung cancer tissues, the level of PD-L1 was positively correlated to the Met-amplification [107,108]. In a microarray assay, inhibiting or knocking down Met substantially reshaped the expression of several immune-related genes, including CD274 [109]. On the contrary, activating Met by hepatocyte growth factor increased PD-L1 expression [109,110].

BRD4
As a member of the bromodomain and extraterminal (BET) family, BRD4 acts as a super-enhancer of oncogenes [111]. In ovarian cancer cells, BET inhibitor reduced PD-L1 expression in a time-and dose-dependent manner [112]. Further, the ChIP assay showed a significant association of CD274 promoter and BRD4 [112]. After BET inhibitor treatment, the associations of CD274 promoter-BRD4 and CD274 promoter-RNA Pol II were decreased, which contributed to the downregulated CD274 transcription [112]. Besides, it was validated that BET inhibitor suppressed CD274 transcription by reducing the BRD4 occupancy at CD274 promoter, independent of c-Myc [113].

Perspectives and conclusion
A growing body of evidence suggests that it is inaccurate to select patients merely by PD-L1 abundance. Understanding the difference between inflammation-induced PD-L1 and oncogenic signal-mediated constitutive PD-L1 is helpful to patient selection. For instance, for EGFR mutant NSCLC patients, α-PD-1 therapy's efficacy was poor despite the high level of PD-L1 [153]. The EGFR mutation-driving NSCLCs commonly harbor lower mutation burdens, and the lower immunogenicity leads to the resistance to α-PD-1 treatments [43]. This oncogenic EGFR-mediated PD-L1 expression could not reflect the real status of the TME. Alternatively, a comprehensive framework containing multiple surrogate markers such as tumor mutation burden would be valuable for selecting patients and predicting outcomes.
Generally, in the TME, the expression of PD-L1 is regulated by numerous factors, including inflammatory stimuli and oncogenic pathways at the levels of transcription, post-transcription, and post-translation.
Exploring potential PD-L1 regulators helps select patients and overcome resistance to α-PD-1/PD-L1 treatments. Besides, the agents regulating PD-L1 expression might be possible adjuvant therapies for the current immune checkpoint inhibitors.

Funding
This work was supported by the National Natural Science Foundation of China (No. 81874120, 82073370).

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Competing interests
The authors declare that they have no competing interests.