Background
Hepatocellular carcinoma (HCC) represents the sixth most common cancer and the fourth leading cause of cancer-related death worldwide [
1]. At least half of HCC patients are diagnosed at advanced stages, precluding potentially curative therapeutic approaches such as hepatectomy or liver transplantation. Immunotherapies that normalize immune responses in the tumor microenvironment have revolutionized the landscape of cancer treatment [
2,
3]. Programmed death-ligand 1 (PD-L1) on tumor cells engages with programmed death 1 (PD-1) on immune cells, resulting in tumor immune evasion [
4]. Therapeutic blockade of the PD-1/PD-L1 immune checkpoint pathways unleashes CD8
+ T cell-mediated immune power against cancer and is a promising clinical anti-tumor therapeutic modality [
5,
6]. In the treatment of advanced HCC, single-agent PD-1 blockade exhibits encouraging survival benefits in early-phase studies, however, the findings are not confirmed in phase III clinical trials [
7,
8]. Hence, understanding the mechanisms underlying PD-1/PD-L1 dysregulation is necessary to improve efficacy of HCC immunotherapy.
Hypoxia is a common characteristic of tumors [
9]. Cellular response to hypoxia has been implicated in several critical aspects of tumor progression, especially anti-cancer immunity [
10]. Hypoxia affects tumor immunity and plays a crucial role in modulating the efficacy of anti-PD-1/PD-L1 treatment in cancers [
11]. In hypoxic tumor cells, the central hypoxia-inducible factor HIF-1α transactivates
CD274, the gene encoding PD-L1 protein, and leads to tumor immune escape from CD8
+ cytotoxic T cells [
12]. Hypoxia causes a HIF-1α-dependent upregulation of PD-L1 and suppresses T cell activation [
13]. Therefore, investigating anti-tumor immunity under hypoxia may help to develop novel approaches for HCC immunotherapeutic strategy.
Circular RNAs (circRNAs) are covalent closed-loop structure without 5’ or 3’ ends and are resistant to RNA exonuclease, providing promising features to serve as potential biomarkers and therapeutic targets [
14]. Fundamental regulatory functions of circRNAs have been demonstrated in various biological processes [
15,
16]. Recent studies illustrate that dysregulation of circRNAs plays an essential role in PD-1/PD-L1-mediated anti-tumor immunity. The circIGF2BP3/PKP3 axis contributes to the immune escape of lung cancer cells through promoting the deubiquitination of PD-L1 [
17]. circBART2.2 promotes the transcription of PD-L1 by binding RIG-I and causes subsequent immune evasion in nasopharyngeal carcinoma [
18]. circDLG1 interacts with miR-141-3p and increases the expression of CXCL12, which promotes gastric cancer resistance to anti-PD-1-based therapy [
19]. Nonetheless, the role of circRNA in HCC immune escape under hypoxic conditions remains obscure.
Herein, we demonstrated that hypoxia-associated circPRDM4 enhanced the immune escape of HCC cells under hypoxia. Mechanistically, circPRDM4 recruited HIF-1α onto the promoter of CD274 and enhanced the HIF-1α-mediated transactivation of the CD274 promoter. Subsequently, circPRDM4 increased tumoral PD-L1 expression level, inhibited CD8+ T cell infiltration, and contributed to immune evasion in HCC.
Materials and methods
Definition of normoxic and hypoxic conditions
Cells were cultured either under normoxic conditions (21% O2, 5% CO2, and 74% N2) or a hypoxic incubator (Memmert GmbH & Co. KG, Schwabach, Germany) providing a hypoxic environment (1% O2, 5% CO2, and 94% N2) for 24 h.
circRNA sequencing
TRIzol reagent (Invitrogen, Carlsbad, CA, USA) was used to extract total RNA. RNA purity and integrity were examined using a NanoDrop ND-1000 spectrophotometer (Thermo Fisher Scientific, Waltham, CA, USA) and an Agilent 2200 TapeStation (Agilent Technologies, Santa Clara, CA, USA), respectively. rRNA was depleted using an Epicentre Ribo-Zero rRNA Removal Kit (Illumina, San Diego, CA, USA), and linear RNA was degraded using RNase R (Epicentre Technologies, Madison, WI, USA). The enrichment protocol following adaptor ligation was performed according to the instructions of the NEBNext Ultra RNA Library Prep Kit for Illumina (NEB, Beverly, MA, USA). The purified RNA was used for cDNA synthesis and sequencing. circRNAs were identified using both CIRCexplorer2 and CIRI2 pipelines. DESeq2 was adopted to determine the differentially expressed circRNAs (> twofold change and P < 0.05).
Quantitative real-time PCR (RT-qPCR)
Total RNA was isolated using TRIzol reagent (Invitrogen). For RNase R treatment, total RNA was incubated with RNase R (3 U/μg; Epicentre Technologies) at 37℃ for 20 min before RT-qPCR detection. Reverse transcription was performed using the PrimeScript RT Master Mix (TaKaRa, Dalian, China). qPCR was performed using TB Green Premix Ex Taq (TaKaRa). The primers for RT-qPCR used in this study are listed in Additional file
1: Table S1. The 2
−ΔΔCt method was used to normalize the data.
Immunofluorescence and fluorescence in situ hybridization (FISH)
Cells or tissue sections were fixed with 4% paraformaldehyde for 30 min, permeabilized with 0.5% Triton X-100 in PBS for 20 min, and then blocked with 5% bovine serum albumin for 1 h. After incubation with the indicated primary antibodies (CD8α, 1:400 dilution, Abcam, Waltham, MA, USA; HIF-1α, 1:400 dilution, Cell Signaling Technology, Danvers, MA, USA) at 4℃ overnight, the cells were washed three times by PBS at room temperature and incubated with the fluorescence-conjugated secondary antibodies for 30 min. Coverslips were mounted on slides using antifade mounting medium. Cell nuclei were stained with 4',6-diamidino-2-phenylindole (DAPI). Images were captured using a fluorescence microscope (Leica Microsystems, Mannheim, Germany).
FAM-labeled FISH probes specific to circPRDM4 (5’-TACACCCTGGCTTTGCGCACAAAC-3’) were designed and synthesized by GenePharma (Shanghai, China). FISH assays were performed using a Fluorescent In Situ Hybridization Kit (GenePharma) according to the manufacturer’s protocols. Briefly, cells were seeded on coverslips, fixed with 4% paraformaldehyde, and incubated with 0.5% Triton X-100 in PBS. After blocking, cells were incubated with FISH probes overnight and washed with SSC washing buffer. Cell nuclei were counterstained using DAPI. A confocal laser scanning microscope (PerkinElmer, Waltham, MA, USA) was used to capture the images.
Nuclear and cytoplasmic fractionation
Subcellular fractionation of cell extracts was performed using the PARIS™ Kit (Invitrogen) following the manufacturer’s protocol. Briefly, the cells were washed using PBS, added with ice-cold cell fractionation buffer, and incubated on ice for 10 min. After the samples were centrifuged at 4℃ and 500 × g for 5 min, the supernatant containing the cytoplasmic fraction were collected for subsequent analyses. The pellet containing the nuclear fraction were added with cell disruption buffer and then collected for further experiments.
Flow cytometry analysis of membrane PD-L1
The cells were centrifuged at 1000 × g for 5 min and collected. After incubating with the PE-conjugated PD-L1 antibody (1:200 dilution; BioLegend, San Diego, CA, USA) in dark for 30 min at 4 °C, cells were resuspended in FACS washing buffer on ice, and subjected to flow cytometry analyses. Samples were obtained and recorded in a FACS Aria II Cell Sorter (BD Biosciences, CA, USA), and data were analyzed with FlowJo software (TreeStar, Ashland, OR, USA).
T cells were activated using anti-CD3 (1 μg/ml) and anti-CD28 (5 μg/ml) antibodies (BD Biosciences, CA, USA) and human recombinant IL-2 (20 ng/ml) for 72 h. HCC cells with circPRDM4 knockdown or overexpression were seeded in plates overnight and incubated with activated T cells for 48 h. The cell culture media was removed. The adherent HCC cells were stained by crystal violent.
Lactate dehydrogenase (LDH) release assay
LDH release assay was performed using a LDH Cytotoxicity Assay Kit (Beyotime, Shanghai, China) according to the manufacturer’s instructions. Briefly, HCC cells were plated in 24-well plates in a density of 4 × 104 cells/well. Activated CD8+ T cells (2 × 105 cells/well) were added at an effector-to-target ratio of 5:1 and cocultured for 16 h. The culture media were centrifuged at 400 × g for 5 min, and the supernatants were transferred into 96-well plates (120 μl/well) and incubated with LDH detection reagent (60 μl/well) for 30 min at room temperature in dark. Absorbance at 490 nm was detected using a microplate reader (Bio-Tek Elx 800; Bio-Tek Instruments, Winooski, VT, USA).
Enzyme linked immunosorbent assay (ELISA)
To measure the levels of TNF-α and IFN-γ produced by cells, the supernatants were collected and the concentrations of TNF-α and IFN-γ were measured by the Human TNF-α ELISA Kit (MultiSciences, Hangzhou, China) and Human IFN-γ ELISA Kit (MultiSciences) according to the manufacturer’s guidelines. Optical densities were determined using a microplate reader (Bio-Tek Elx 800; Bio-Tek Instruments) at 450 nm.
Animal experiments
For patient-derived xenograft (PDX) mouse model, 2 × 106 patient-derived primary HCC cells mixed with Matrigel (Corning Inc., Corning, NY, USA) were subcutaneously transplanted in NCG mice. Human HCC tissues were minced into small pieces, treated with DNase I (20 μg/ml) and collagenase type II (100 U/ml in HBSS) for 30 min at 37 °C. Supernatants were collected and filtered through Falcon 70 μm Cell Strainer (Corning Inc.). The T cell fraction was collected using a prepared Percoll (GE Healthcare, Uppsala, Sweden) gradient by centrifuging at 800 × g for 30 min. Human CD8+ T cells were sorted from tumor infiltrating lymphocytes (TILs) by flow cytometer. CD8+ T cells were activated using anti-CD3 (1 μg/ml) and anti-CD28 (5 μg/ml) antibodies and human recombinant IL-2 (20 ng/ml) for 72 h. Each mouse was injected with 2 × 106 of CD8+ T cells via caudal vein to reconstitute the human immune system. At day 7 after injecting with CD8+ T cells, circPRDM4 plasmid, empty vector plasmid, cholesterol-conjugated si-circPRDM4, or the negative control RNAi was intratumorally injected every three days for three weeks. Tumor growth was measured regularly. At day 28, mice were sacrificed and tumors were weighed and subjected for subsequent RT-qPCR and immunofluorescence analyses. To investigate whether knockdown or overexpression of circPRDM4 affect the proliferation of HCC cells under immunodeficient conditions, we also constructed the aforementioned PDX mouse models without adding immune cells to the mice. After intratumorally injection with circPRDM4 plasmid, empty vector plasmid, cholesterol-conjugated si-circPRDM4, or the negative control RNAi every three days for three weeks, the tumor growth and weight were compared between different groups.
Animal experiments were performed in compliance with the Animal Research: Reporting In Vivo Experiments (ARRIVE) guidelines and approved by the Institutional Animal Care and Use Committee of Nanjing Medical University (No. 10791) and the Animal Welfare and Ethical Committee of Wannan Medical College (No. LLSC-2020-090).
RNA pull-down and mass spectrometry
RNA pull-down assay was performed using a Magnetic RNA–protein Pull-down Kit (Thermo Fisher Scientific). Briefly, cell lysates were incubated with biotin-labeled probes (RiboBio, Guangzhou, China) and streptavidin magnetic beads (Invitrogen). After incubation at 4℃ overnight, beads were separated magnetically and washed five times. For mass spectrometry analysis, the beads were incubated with ddH2O at 70℃ for 5 min. For western blotting, the beads were boiled in SDS for protein elution.
RNA immunoprecipitation (RIP) assays
RIP assays were carried out using a Magna RIP RNA-Binding Protein Immunoprecipitation Kit (EMD Millipore, Billarica, MA, USA). Briefly, the cells were washed twice with ice-cold PBS, transferred to centrifuge tubes, and collected by centrifugation at 1500 rpm for 5 min at 4℃. Cells (1 × 107) were resuspended in RIP Lysis Buffer and incubated on ice for 5 min. To confirm the binding between circPRDM4 and HIF-1α, 50 μl of beads coated with anti-HIF-1α antibody (5 μg; Cell Signaling Technology) or IgG antibody (5 μg; EMD Millipore) were incubated with the cell lysates in RIP Immunoprecipitation Buffer with rotating at 4℃ overnight. The protein-RNA complexes were washed six times with cold RIP Wash Buffer. The enriched RNA was purified and then isolated using TRIzol reagent and analyzed by RT-qPCR. To determine the HIF-1α domain required for circPRDM4 binding, cells were transfected with Flag-tagged bHLH (basic helix-loop-helix), PAS (Per-ARNT-Sim), or TAD (transactivation domain) truncated variants of HIF-1α, which were generated by GeneCreate (Wuhan, China). Cells were lysed and then incubated with anti-Flag antibody (5 μg; Cell Signaling Technology) or IgG antibody (5 μg; EMD Millipore) at 4 °C overnight. The abundance of protein-bound RNA was examined using RT-qPCR.
Chromatin immunoprecipitation (ChIP) and chromatin isolation by RNA purification (ChIRP) assays
ChIP assays were performed using the Pierce Magnetic ChIP Kit (Thermo Fisher Scientific). Briefly, cells were crosslinked with 1% formaldehyde for 10 min at room temperature. Glycine was then added and incubated with the cells for 5 min to quench the crosslinking reaction. After washing twice with PBS, the cells were lysed in lysis buffer including protease and phosphatase inhibitors. Lysates were sonicated to shear DNA into smaller and workable pieces. Chromatin was incubated with anti-HIF-1α antibody (1:100 dilution; Cell Signaling Technology) or IgG antibody (2 μg; EMD Millipore) overnight at 4 °C. The following day, protein A/G magnetic beads were added and incubated for 2 h at 4 °C. After washing steps, DNA was eluted from beads and purified. The precipitated DNA was analyzed by qPCR or processed for ChIP-seq. CD274 promoter-specific primers used for qPCR were as follows: CD274 (− 2000 to − 1500 bp): Forward: 5’-ACACGAATCCTCACATTACT-3’; Reverse: 5’-AATCATATCCTCCTAGATGGC-3’; CD274 (− 1500 to − 1000 bp):Forward: 5’-TTCGGGAACTTTGGGAAG-3’; Reverse: 5’-GCTGACACTGCCTTGATT-3’; CD274 (− 1000 to − 500 bp):Forward: 5’-ATTATGACACCATCGTCTGT-3’; Reverse: 5’-TCGTGGATTCTGTGACTTC-3’; CD274 (− 500 to 0 bp):Forward: 5’-CAGATGTTGGCTTGTTGTAA-3’; Reverse: 5’-GTATCTAGTGTTGGTGTCCTA-3’.
For ChIRP assays, 4 × 107 cells were harvested and washed with PBS twice. Cells were fixed in 1% formaldehyde in PBS for 10 min at room temperature. Crosslinking was quenched with glycine for 5 min. After centrifuging at 1500 × g for 5 min at 4 ℃, cells were washed by ice-cold PBS twice. Crosslinked cell pellets were resuspended in lysis buffer and sonicated to produce chromatin fractions. circPRDM4 probe was designed at the junction site. All probes were synthesized with BiotinTEG at the 3’ end. Lysates were incubated with probes at 37 °C for 4 h. Streptavidin beads were then added to isolate probe binding complex. After washing steps, the bound DNA was quantified with RT-qPCR with CD274 promoter-specific primers or processed for sequencing.
Dual-luciferase reporter assay
The CD274 wild-type or mutant promoter region was fused to the promoterless firefly luciferase gene of pGL3-Basic vector (Promega, Madison, MI, USA), and cells were transfected with luciferase reporter plasmid, the HIF-1α plasmid, the circPRDM4 plasmid, circPRDM4 truncated fragments, or the corresponding control plasmids. The luciferase activity of the cells was evaluated after 48 h using the Dual-Luciferase Reporter Assay System (Promega) according to the manufacturer’s instructions. The measurements were normalized to the ratio between firefly activity and renilla luciferase activity.
Dot blotting
For dot blotting, wild-type or mutant circPRDM4 linearized RNAs were dropped onto Hybond-N+ membrane (Cytiva, Marlborough, MA, USA) followed by ultraviolet crosslinking. RNA signals were then detected using biotin-labeled single-stranded DNA segment of CD274 promoter. Biotin signals were detected with HRP-conjugated streptavidin according to the manufacturer's instructions (Thermo Fisher Scientific).
DNA in situ hybridization
The cells were treated with 0.1% NP-40 in 2 × SSC for 30 min, followed by digestion in proteinase K in TBS for 10 min at 37 °C. Serial dehydration was with ethanol to a final concentration of 100%. Cells were denatured for 8 min at 75 °C and replaced with ethanol to a final concentration of 100%. Afterwards, cells were air-dried and incubated with probes for CD274 promoter and hybridization buffer overnight at 37 °C. The following day, cells were washed three times, 5 min each time: first with preheated 2 × SSC at 53 °C, then with preheated 2 × SSC with 0.1%NP-40 at 42 °C, and lastly with preheated 2 × SSC at 42 °C. Cell nuclei were stained with DAPI in dark for 10 min.
Immunoprecipitation (IP)
For IP between HIF-1α and p300/CBP, cells were dissolved in RIPA, added with beads, mixed with anti-HIF-1α antibody (5 μg; Cell Signaling Technology) or IgG antibody (5 μg; EMD Millipore) at 4 °C overnight. The next day, the immunoprecipitated proteins were added with SDS-PAGE loading buffer, heated at 95 °C for 10 min, and subjected to western blotting. The dilution for anti-p300 antibody (Abcam) and anti-CBP antibody (Abcam) was 1:1000.
Statistical analysis
Comparisons between groups were assessed using two-sided Student’s t test. Correlations between groups were evaluated using the Pearson’s correlation coefficient or Spearman’s ρ when appropriate. The significance of Kaplan–Meier survival curves was estimated with the log-rank test. All statistical analyses were performed using SPSS 26.0 (IBM Corporation, Armonk, NY, USA) or GraphPad Prism 9.0 (GraphPad Software, La Jolla, CA, USA), setting statistical significance at P < 0.05. *P < 0.05, **P < 0.01, ***P < 0.001, and “ns” stands for “no significance”.
Additional methodological details are included in Additional file
2: Supplementary Materials and Methods.
Discussion
Blockade of immune checkpoints contributes to HCC regression, however, the response rate remains quite low [
22]. Understanding the mechanisms underlying immune escape is necessary to achieve better survival outcomes in advanced HCC patients in the clinical context [
23]. In this study, we found that circPRDM4 was highly expressed in responders for anti-PD-1 therapy. circPRDM4 expression level was correlated to the therapeutic efficacy of PD-1 blockade. Improved PFS and OS were observed in PD-1 blockade-treated HCC patients with high circPRDM4 expression. In addition, circPRDM4 upregulation was associated with less CD8
+ cell infiltration and high
CD274 expression level.
CD274 mRNA regulation is an intricate process, which involves genomic alterations, epigenetic modification, transcriptional regulation, and post-transcriptional modification [
4]. Future studies with larger sample size are warranted to confirm the correlation between circPRDM4 and
CD274 mRNA level and to piece together the puzzle of
CD274 regulation network.
Hypoxia has been acknowledged as a potent factor that impedes anti-tumor immunity by reshaping the tumor microenvironment [
24]. HIF-1α is a core regulator under hypoxic conditions, and has been proved as a direct regulator of PD-L1 [
12,
13]. We performed ChIP and dual-luciferase reporter assays and confirmed that HIF-1α bound to
CD274 promoter and transactivated PD-L1. Therefore, tumor cells may leverage the HIF-1α/PD-L1 axis to evade from immune surveillance. Reportedly, SLC7A11 upregulates PD-L1 expression through the HIF-1α cascade and contributes to the formation of an immunosuppressive microenvironment, thus promoting HCC metastasis [
21]. In glioma cells, the direct binding between HIF-1α and
CD274 promoter region results in elevated PD-L1 expression under hypoxia [
25]. Here, we indicated that circPRDM4 expression level was increased under hypoxia conditions in HCC cells. Since expression of PD-L1 is frequently detected in human malignancies, PD-L1 expression on cancer cells and other cells in the tumor microenvironment is of major clinical relevance [
26]. In the present study, we focused on the effects of circPRDM4 on PD-L1 expression in HCC cells. We performed loss- and gain-of function experiments, and showed that circPRDM4 inhibited CD8
+ T cell-mediated anti-tumor immunity mainly through upregulating PD-L1. Results from RNA pull-down, mass spectrometry, and RIP assays indicated that circPRDM4 could interact with HIF-1α. Rescue experiments verified that circPRDM4 facilitated immune escape of HCC cells mainly by the HIF-1α/PD-L1 axis.
Mechanistically, our work revealed that circPRDM4 not only bound to the bHLH domain of HIF-1α but also the promoter of
CD274. The bHLH domain defines a large superfamily of eukaryotic transcription factors and mediates the combination of HIF-1α and DNA [
20]. We therefore investigated the role of circPRDM4 in the interaction between HIF-1α and
CD274 promoter. Intriguingly, we found that circPRDM4 could function as a scaffold to recruit HIF-1α onto
CD274 promoter, and cemented their interaction, ultimately promoting the HIF-1α-mediated transactivation of PD-L1. Knockdown of circPRDM4 impeded the interaction between HIF-1α and
CD274 promoter, and dampened the expression level of PD-L1. Recently, emerging evidence has revealed that circRNAs can serve as scaffold to recruit proteins, especially transcription factors, to chromatin [
27]. circIPO11 recruits TOP1 to
GLI1 promoter to activate its transcription, and drives self-renewal of liver cancer initiating cells [
28]. circKcnt2 recruits the NuRD complex to inhibit
Batf transcription, thus facilitating colitis resolution [
29]. In prostate cancer, circ0005276 interacts with FUS so as to initiate the transcription of
XIAP [
30]. Upregulated circAnks1a in spinal cord increases YBX1 recruitment to
Vegfb promoter, thereby triggering its transcription [
31]. In HCC, circRHOT1 recruits TIP60 to the promoter region of
NR2F6 and facilitates its transcription [
32]. circPOK interacts with ILF2/3 complex to promote
Il6 transcription in mesenchymal tumors [
33]. Our work demonstrated that the circPRDM4-HIF-1α-
CD274 ternary complex reinforced the interaction of HIF-1α with
CD274 promoter. This interaction was at least in part required for the tumor-promoting roles of circPRDM4 in immune evasion of HCC cells under hypoxic conditions.
As reported by various clinical trials, among the patients treated with anti-PD-1 monotherapy, PD-L1 positive HCCs respond better than those with negative PD-L1 expression [
34‐
36]. Genomic analyses for the GO30140 phase Ib and IMbrave150 phase 3 trial of atezolizumab and bevacizumab in patients with HCC reported that high expression of PD-L1, as per RNA-seq, is related to better response and longer PFS [
37]. A preclinical study by Liu et al
. reported that ADORA1 expression is negatively correlated with PD-L1 expression, and patients with low ADORA1 expression show tumor shrinkage after PD-1 mAb treatment [
38]. In the present study, we found that circPRDM4 induced PD-L1 expression, and circPRDM4 was related to better response to PD-1 blockade.
In conclusion, hypoxia-associated circPRDM4 promoted PD-L1 transcription and contributed to HCC cell evading from CD8+ T cell-mediated anti-tumor immunity. To our knowledge, the present work reveals the first evidence showing a circRNA acting as a scaffold in HIF-1α-mediated immune escape. The roles of circPRDM4 in hypoxic immunosuppressive microenvironment not only present novel insight into immune escape of tumor cells, but also provide a novel prognostic biomarker and therapeutic candidate for cancer immunotherapy.
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