Introduction
Cholangiocarcinoma (CCA) is the second most common primary liver malignancy that accounts for approximately 15% of cases of liver cancers and ~ 3% of all gastro-intestinal malignancies [
1,
2]. In recent years, the morbidity and mortality of CCA have been steadily rising. Anatomically, CCA can be classified as intrahepatic (iCCA), perihilar, or distal cholangiocarcinoma [
3]. There are limited treatment options for CCA, which is a lethal malignancy with a five-year overall survival (OS) rate of only ~ 10% and a median OS of ~ 24 months [
4]. Currently, surgical resection and liver transplantation are the only curative treatment approaches used in early-stage patients. However, once diagnosed, the iCCA patients are generally in the advanced stages of the disease when an effective surgical treatment is no longer possible. Moreover, present therapeutic strategies have a limited efficacy. Therefore, effective therapeutic strategies and potential molecular mechanisms to combat CCA are necessary.
Numerous studies have unveiled
KRAS, BRAF, IDH1/2, FGFR2, and
EGFR as prevalent oncogenic drivers in CCA [
5], especially the
KRAS mutations, which might be one of the most frequent molecular alterations in iCCA [
6]. Once KRAS is activated, the Raf/MEK/ERK pathway is hyper-activated, thus, modulating multiple cellular processes, including survival, proliferation, and differentiation. Therefore, plentiful research studies have been dedicated to study this pathway’s inhibition, including using MEK1/2 and ERK1/2 inhibitors [
7‐
9]. Even though MEK inhibitors, such as trametinib, have been extensively investigated, whether ERK inhibitors can be used for the treatment of iCCA, especially for
KRAS-mutated iCCA, has not been properly evaluated in vivo or in vitro.
A wide spectrum of cancers, including iCCA, have been treated using the immune checkpoint blockade (ICB) therapy. As the most crucial immune checkpoint, the PD-L1 expression level determines the efficacy of anti-PD-1/PD-L1 immunotherapy. Accumulating evidence has suggested that KRAS can regulate PD-L1 expression in other cancers, such as NSCLC [
10]. However, whether PD-L1 can be modulated by ERK in iCCA and the underlying molecular mechanisms remain unclear. In addition, as a highly conserved intracellular material degradation process, autophagy is involved in immune responses and cell homeostasis maintenance [
11]. A recent study has discovered that autophagy regulates PD-L1 expression in gastric cancer [
12]. Consequently, the link between ERK signaling, PD-L1, and autophagy in iCCA needs to be determined.
The present study uncovered an underlying molecular mechanism for PD-L1 regulation by ERK signaling via autophagy activation and provided a novel immune-related therapeutic strategy that regulates the ERK signaling pathway.
Materials and methods
Cell lines and cell culture
Human iCCA cell lines HuCCT1 and RBE were purchased from the Cell Bank of Type Culture Collection of Chinese Academy of Sciences (Shanghai, China) and maintained in RPMI 1640 medium (Sigma-Aldrich, MO, USA) supplemented with 10% heat-inactivated fetal bovine serum (Gibco, USA), 100 U/mL penicillin, and 100 mg/mL streptomycin (Sigma-Aldrich). All cell lines were cultured at 37 °C in a humidified incubator with 5% CO2.
Antibodies and reagents
The antibodies used in the present study included the following: anti-SQSTM1/p62 rabbit mAb (ab109012) from Abcam, anti-LC3A/B (12741), anti-ATG7 (8558), anti-PD-L1 (13684 or 41726), anti-KRAS (3339), anti-p44/42 MAPK (Erk1/2) (4695), anti-Beclin-1(3495), anti-ATG5 (12994), and anti-phospho-p44/42 MAPK (Erk1/2) (Thr202/Tyr204) (4370) from Cell Signaling Technology, and anti-GAPDH from Yeasen. Annexin V-APC and 7-AAD apoptosis detection kit were purchased from Biolegend. Trametinib and SCH772984 were purchased from Selleck Chemicals (Houston, TX, USA).
Western blotting analysis
Western blotting analysis was performed as previously described [
13]. Briefly, the proteins from total cell lysates were separated using 10% or 12.5% standard sodium dodecyl sulfate polyacrylamide gels and then transferred to polyvinylidene difluoride membranes. The membranes were washed, blocked, and incubated at 4 °C overnight with specific primary antibodies against Beclin-1, ATG5, ATG7, LC3, ERK, p-ERK, KRAS or PD-L1 (1:1000), P62(1:7000), and GAPDH (1:5000), followed by incubation with horseradish peroxidase (HRP)–conjugated secondary antibodies. The intensity of protein bands was determined using densitometry with a Bio-Rad system (Bio-Rad Laboratories, CA, USA).
RNA interference
Three different sequences targeted to three different sites in KRAS and PD-L1 were designed by Genechem (Shanghai, China). Sense and antisense strands for siRNAs were as follows: siKRAS-1: 5’-CGAAUAUGAUCCAACAAUATT-3’; 5’-UAUUGUUGGAUCAUAUUCGTT-3’; siKRAS-2: 5’-AGCAAGAAGUUAUGGAAUUTT-3’;5’-AAUUCCAUAACUUCUUGC-UTT-3’; siKRAS-2: 5’-CAAGAGGAGUACAGUGCAATT-3’; 5’-UUGCACUGUACUC-CUCUUGTT-3’; siPD-L1: 5’-GACCUAUAUGUGGUAGAGUAU-3’;5’-AUACUCUACCACAUAUAGGUC-3’; KRAS and PD-L1siRNAs were transfected into iCCA cells using Lipofectamine 3000 (Invitrogen, USA). Cells were lysed 72 h after transfection, and proteins levels were assayed using western blotting analysis.
Tissue microarray (TMA) and immunohistochemistry analysis
TMA was constructed as previously described [
14]. In summary, all patients diagnosed with hepatocellular carcinoma were reviewed by two histopathologists, and representative areas were pre-marked in the paraffin blocks away from necrotic and hemorrhagic materials. Duplicates of 1-mm-diameter core biopsies from tumor center and para-tumoral noncancerous area were taken from the donor tumor tissues and transferred to the defined array positions. Thus, a TMA block containing 184 cylinders was constructed (Shanghai Biochip Co., Ltd.). Sections (4 μm in thickness) were placed on 3-aminopropyltriethoxysilane–coated slides. Immunohistochemistry was performed with monoclonal rabbit antibodies against human ERK (1:250), p-ERK (1:400), and PD-L1 (1:200) using 4-μm-thick paraffin-embedded tumor tissue sections excised from patients diagnosed with iCCA at the Liver Cancer Institute, Zhongshan Hospital, Fudan University, Shanghai, China. After deparaffinization and rehydration, antigen retrieval was performed using pepsin (10 μM) for 20 min at 37 °C and by boiling the slides in citrate buffer (pH 6.0) for 10 min in a microwave for all other antigens. Cooled slides were washed in phosphate-buffered saline (PBS; 3 × 15 min each) and incubated in 3% hydrogen peroxide for 10 min to block endogenous peroxidase activity. Following washing, slides were blocked with 10% goat serum for 1 h at room temperature and probed with primary antibodies overnight at 4 °C in a humid chamber. Slides were then incubated with HRP-conjugated secondary antibodies for 1 h at room temperature. After washing, slides were developed using a diaminobenzidine substrate kit (Gene Tech, Shanghai, China), counterstained with hematoxylin for 2 min, washed, and dehydrated.
Patients and specimens
Patient samples were collected after obtaining informed consent from each patient according to an established protocol approved by the Ethics Committee of Zhongshan Hospital. The data did not contain any information that could lead to identification of individual patients.
Tumor specimens for TMA studies were obtained from 92 consecutive iCCA patients who underwent curative resection without preoperative treatment at the Liver Cancer Institute, Zhongshan Hospital, Fudan University. Samples were collected immediately after resection, transported in liquid nitrogen, and stored at −80 ℃. An additional 11 fresh tissue samples were also obtained for western blotting.
Flow cytometry
Activated T cells were added into the co-culture system with HuCCT1 or RBE cells treated with CQ or transfected with shATG7 at a ratio of 1:1, respectively. After 24 h, suspended T cells and the trypsinized HuCCT1 or RBE cells were removed from the cell culture plate. Then, after washing with PBS, the suspended cells were stained with Annexin V-APC and 7-AAD for 15 min with an apoptosis detection kit at room temperature (Biolegend, San Diego, CA, USA). Finally, the apoptotic cells were evaluated using flow cytometry (BD FACSAriaTM II, NJ, USA) and the data were analyzed using FlowJo software according to the manufacturer’s instructions.
Immunofluorescence and confocal microscopy
For immunofluorescence analysis, cells were fixed with 4% paraformaldehyde for 15 min, permeabilized in PBS containing 0.1% Triton X-100 for 10 min, and then blocked with 5% bovine serum albumin for 20 min. Slides were incubated with the indicated primary antibodies overnight at 4 °C, followed by incubation with Alexa Fluor 488 secondary antibody for 1 h at room temperature. Then, 4’,6-diamidino-2-phenylindole was used to stain the nuclei and images were acquired using confocal microscopy (Olympus FV-3000, Japan).
Cell counting kit-8 assay
Cellular apoptosis rate was detected using a cell counting kit-8 (CCK-8) (Dojindo, Japan) according to the manufacturer’s instructions. The indicated cells were inoculated into 96-well plates. At the indicated point, 10 μL of the CCK-8 solution were added into each well with 100 µL of culture media. The absorbance of each individual well was measured at 450 nm.
Peripheral blood mononuclear cells isolated from heparinized peripheral blood samples were obtained from healthy donors using Ficoll-Paque (GE Healthcare Life Sciences) density gradient centrifugation. They were cultured in ImmunoCult™-XF T Cell Expansion Medium (STEMCELL Technologies, Canada) with ImmunoCult Human CD3/CD28 T cell activator (STE-MCELL Technologies) and IL-2 (600 U/mL; PeproTech, USA) for one week according to the manufacturer’s protocol. Cancer cells were seeded in the plates overnight and then incubated with CQ (5 or 10 μM) for 24 h, followed by incubation with activated T cells for 24 h. The ratio between cancer cells and activated T cells was 1:1. T cells and cell debris were removed using a PBS wash and the remaining cells were stained with crystal violet or used in subsequent experiments.
Statistical analysis
Differences between two groups were evaluated using paired or unpaired two-tailed Student's t-test, while multiple groups were analyzed using one-way analysis of variance. Correlation analyses were used to analyze the relationships among the expression levels of ERK, pERK, and PD-L1. Representative results from three independent experiments were used in the study analysis. Statistical significance was set at P < 0.05. All analyses were performed using SPSS version 26.0 software (IBM SPSS Inc., USA).
Discussion
A high prevalence of oncogene
KRAS mutations have been frequently observed in a variety of cancers, including human iCCA [
19,
20]. In general, KRAS gene mutation at different prevalence rates is associated with a series of highly fatal cancers. The top 5 human cancers among these KRAS somatic mutations are pancreas (57%), large intestine (35%), biliary tract (28%), small intestine (17%) and lung (16%) [
21]. Apparently, the mutation sites and frequencies of KRAS gene were varied from diverse human tumor types. For example, three amino acid residues with missense mutations including G12(codon 12), G13(codon 13), and Q61(codon 61) are the most common mutation sites with distinct mutation frequencies in different cancers [
22]. Among these mutations, point mutations in KRAS codon 12 are probably the most common event, which accounts for approximately 80% of KRAS mutation [
21]. A recent study also showed that, compared with a western iCCA cohort (the Memorial Sloan Kettering Cancer Center cohort), the China Fudan-iCCA cohort showed higher KRAS mutation frequency and lower IDH1, ARID1A, and TERT mutation frequencies [
23]. Most studies have concluded that there is a negative influence on prognosis of patients with a
KRAS mutation whether or not they have undergone surgery for other solid tumors [
24,
25] and a correlation between oncogenic
KRAS mutations and PD-L1 expression in cancers has also been emphasized [
10,
26,
27]. As a major downstream part of KRAS signaling, the Ras/Raf/MEK/ERK pathway plays a pivotal role in tumor initiation, progression, and differentiation. Nevertheless, the mechanism of KRAS with its downstream pathways in modulating PD-L1 expression remained uncertain. The present study suggested that RNA interference in
KRAS down-regulates the expression of PD-L1 via ERK signaling in an autophagy-dependent manner as illustrated above. Based on this foundation, it was speculated that the poor prognosis of
KRAS-mutated patients is due to the continuous activation of the Ras/Raf/MEK/ERK pathway caused by the
KRAS gene mutation, which might lead to up-regulation of PD-L1 in tumor cells by abrogating autophagic degradation and further promoting the immune escape.
To investigate the effects of ERK inhibition on PD-L1 expression in
KRAS-mutated iCCA cells, HuCCT1 and RBE cells were treated with MEKi and ERKi for 24 h. The results demonstrated that the inhibition of MEK by trametinib and ERK by SCH772984 promoted the degradation of PD-L1, but not in a dose-dependent manner. In addition, these results showed that there was little change in the expression of ERK after using ERKi, while ERK phosphorylation and PD-L1 level were altered significantly, suggesting that it was functional phosphorylated ERK that modulated the expression of PD-L1. Immunohistochemical score and western blotting results also showed a positive association between p-ERK and PD-L1 (Fig.
5B, D and E). In line with the present findings, mounting evidence has pointed to a key role of ERK signaling in modulating the expression of PD-L1. Upon activation, ERK signaling reinforced the binding of c-JUN and PD-L1 promoters, which further recruited STAT3 to increase PD-L1 expression in NSCLC [
28]. Furthermore, it has also been reported that PD-L1 expression induced by TRAIL was dependent on the p-ERK/STAT3 signaling pathway in esophageal squamous carcinoma [
29]. Except for ERK signaling, the PI3K/AKT, JAK/STAT3, and Wnt/β-catenin signaling pathways were also involved in the modulation of PD-L1 expression in various solid tumors [
30‐
32]. In general, these studies and the present results have demonstrated that it was the ERK signaling pathway, or more precisely the functional p-ERK, that mainly modulated the expression of PD-L1.
Previous studies have reported that trametinib promotes the autophagic flux in RAS-driven cancers [
18]. As the downstream component of the Ras pathway, it was hypothesized that inhibition of ERK may have the same effect, which was also confirmed by the present study experiments. These findings showed that HuCCT1 and RBE cells treated with ERK inhibitor did not only reduce the expression of PD-L1, but also induced degradation of p62, conversion of LC3-I to LC3-II, and increase in LC3 puncta in the cytoplasm, which indicated the autophagy activation. In line with the findings that autophagy inhibition enhances PD-L1 expression in gastric cancer [
12], the present research showed for the first time that ERK inhibition partially degraded PD-L1 via the autophagy pathway. Both pharmacologically inhibiting autophagy and genetically silencing
ATG7 partially reversed the decrease in PD-L1 expression caused by the ERK inhibitor (Fig.
3). Consequently, the promoted autophagic flux was part of the PD-L1 down-regulation mechanism due to ERK inhibition. However, restriction of autophagy alone by CQ or knocking down
ATG7 did not significantly alter the PD-L1 expression, which meant that the autophagy pathway, which takes place downstream of p-ERK, is involved in the regulation of PD-L1 expression. Nevertheless, whether the alteration of PD-L1 modulates autophagy after ERK inhibition needs further exploration.
Undoubtedly, PD-1/PD-L1-associated immune escape remains the principal cause of a number of patients’ failure to achieve a durable response to PD-1/PD-L1 blockade [
33]. As an important mechanism for tumor cells to evade immune surveillance, PD-L1 expressed on tumor cells prevented T cells from effectively identifying and eradicating them [
34]. The immunohistochemical analysis also showed that compared to para-tumor tissues, PD-L1 staining intensity was predominantly higher in tumor tissues. Due to the binding between PD-1 on T cells and PD-L1 on tumor cells, T cells infiltrating the tumor microenvironment, which are probably the most potent weapon for tumor elimination [
35], were dysfunctional and exhausted [
36], leading to a reduction in apoptosis of cytotoxic T cell-mediated tumor cells. In view of this point, our results indicate that upon MEKi or ERKi treatment, the level of PD-L1 expression was visibly down-regulated. More importantly, the apoptosis rate in HuCCT1 or RBE cells pretreated with ERKi was enhanced after co-culture with CD3/CD28-activated CD8
+T cells compared to that in the control group, which was manifested by an increase in tumor cell apoptosis rate detected by the CCK-8 assay or flow cytometry. Based on these results, it is feasible that blocking ERK phosphorylation might abrogate PD-L1-mediated immunosuppression and promote the infiltration of functional cytotoxic T lymphocytes in solid tumors. Thus, considering the anti-tumor effect of ERK inhibitor and its regulatory role in tumor microenvironment, we reasoned that a combination treatment of ERK-targeted therapy and anti-PD-1/PD-L1 immunotherapy might block interactions between PD-1/PD-L1 pathway molecules more completely and restore CD8
+T cell recognition in tumor cells to enhance the T cell-mediated immune response and anti-tumor activity. A recent study has also reported that the ERK pathway inhibitor in combination with anti–PD-1 monoclonal antibody suppresses tumor growth and improves survival in mice [
37].
In summary, the present study elucidated a novel mechanism by which ERK signaling regulates the PD-L1 expression via the autophagy degradation pathway and enhances the T cell-mediated immune response. These findings shed light on the clinical application of ERK-targeted therapy together with anti-PD-1/PD-L1 immunotherapy, which represents a promising strategy for RAS-mutated iCCA treatment.
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