The miR-23a/27a/24-2 cluster drives immune evasion and resistance to PD- 1/PD-L1 blockade in non-small cell lung cancer

doi:10.21203/rs.3.rs-5028588/v1

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The miR-23a/27a/24-2 cluster drives immune evasion and resistance to PD- 1/PD-L1 blockade in non-small cell lung cancer

https://doi.org/10.21203/rs.3.rs-5028588/v1

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Programmed cell death protein ligand-1 (PD-L1) and major histocompatibility complex Ⅰ (MHC-Ⅰ) are key molecules related to tumor immune evasion and resistance to programmed cell death protein 1 (PD-1)/PD-L1 blockade. Here, we demonstrated that upregulation of all miRNAs in miR-23a/27a/24 − 2 cluster correlated with poor survival, immune evasion and PD-1/PD-L1 blockade resistance in non-small cell lung cancer (NSCLC). Overexpression all miRNAs in miR-23a/27a/24 − 2 cluster upregulated PD-L1 by targeting Cbl proto-oncogene B and downregulated MHC-Ⅰ through increasing eukaryotic initiation factor 3B (eIF3B) by targeting microphthalmia associated transcription factor. In addition, we demonstrated that miR-23a/27a/24 − 2 cluster miRNAs maintain its expression in NSCLC through increasing Wnt/β-catenin signaling regulated interaction of transcription factor 4 (TCF4) and miR-23a/27a/24 − 2 cluster promoter. Notably, pharmacologic targeting eIF3B pathway dramatically enhanced PD-1/PD-L1 blockade sensitivity in miRNAs of miR-23a/27a/24 − 2 cluster high expressing NSCLC by upregulating MHC-Ⅰ expression and keeping miR-23a/27a/24 − 2 cluster-induced high expression of PD-L1. In summary, we elucidate the mechanism by which miR-23a/27a/24 − 2 cluster miRNAs maintain its own expression and the molecular mechanism by which miR-23a/27a/24 − 2 cluster miRNAs promote tumor immune evasion and PD-1/PD-L1 blockade resistance. In addition, we provide a novel strategy for treatment of miR-23a/27a/24 − 2 cluster high expressing NSCLC.

Lung cancer is the most common malignancy and the leading cause of cancer death in worldwide [1]. Non-small cell lung cancer (NSCLC) is the most common type of lung cancer, account for 85% of lung cancer cases [2]. Despite improvements in therapy in recent years, the mortality of NSCLC patients is still high, with less than 20% of patients surviving after 5 years [2, 3]. Growing evidences showing that immune evasion is an important factor leading to cancer progression and a major stumbling block in effective anticancer therapy [4]. Thus, immunotherapy such as immune checkpoint blockade (ICB) has become an important therapeutic intervention for a range of cancer types, including NSCLC, and observed promising results [3, 5]. However, either ICB alone or combination with chemotherapy, only a subset of cancer patients has achieved long-term remission [3, 6]. Therefore, more clearly understanding the mechanisms of tumor immune evasion and resistance to ICB is critical for development of future therapies.

Studies have shown that many factors involved in the regulation of tumor immune evasion, including microRNAs (miRNAs). Notably, miRNA-cluster synergistically affects the tumor immune evasion through that different members of miRNA-cluster simultaneously target the same gene or different genes in the same signaling pathway [7, 8]. The miR-23a/27a/24 − 2 cluster includes miR-23a, miR-27a, and miR-24-2, and our previous study has shown that miRNAs of miR-23a/27a/24 − 2 cluster is upregulated in early stage NSCLC and promotes postoperative recurrence through activating Wnt/β-catenin signaling [9]. Importantly, activation of Wnt/β-catenin signaling associated with immune escape and resistance to ICB treatment in NSCLC [10]. In addition, studies show that miR-23a/27a/24 − 2 cluster can suppress interferon-γ (IFN-γ) expression and antigen-specific cytotoxicity in CD8⁺ T cells [11]. Based these findings, we hypothesize that upregulated miRNAs of miR-23a/27a/24 − 2 cluster may be involved in immune evasion and ICB resistance in NSCLC.

In this study, we demonstrated that upregulated expression of miR-23a/27a/24 − 2 cluster miRNAs was closely associated with immune evasion and PD-1/PD-L1 blockade therapeutic resistance in NSCLC. Our data show that miRNAs of miR-23a/27a/24 − 2 cluster significantly inhibited IFN-γ secretion of T cells, suppressed the infiltration of CD8⁺ T cells into tumor tissues, downregulated major histocompatibility complex class Ⅰ (MHC-Ⅰ) expression and upregulated programmed cell death ligand 1 (PD-L1) expression in NSCLC. Mechanistically, miRNAs of miR-23a/27a/24 − 2 cluster upregulates PD-L1 expression by targeting PD-L1 negative regulator Cbl proto-oncogene B (CBLB), and downregulates MHC-Ⅰ through upregulating eukaryotic initiation factor 3B (eIF3B) expression by targeting eIF3B negative regulator microphthalmia transcription factor (MITF) in NSCLC. Further, we identified that miR-23a/27a/24 − 2 cluster miRNAs maintain its expression by increasing the interaction of transcription factor 4 (TCF4) and miR-23a/27a/24 − 2 cluster promoter through activating Wnt/β-catenin signaling. Finally, we demonstrated that pharmacologic targeting eIF3B pathway or β-catenin/TCF4 axis can improve the efficacy of PD-1/PD-L1 blockade in NSCLC with high expression of miRNAs in miR-23a/27a/24 − 2 cluster. Notably, targeting eIF3B more effectively promoted the therapeutic efficacy of PD-1/PD-L1 blockade.

Human specimens and Cell culture

NSCLC specimens were collected from 82 patients with NSCLC during surgery or by biopsy (Table S1) at Daping Hospital under a protocol approved by the ethical review committees. All cell lines used in this study was purchased from Shanghai Cell Bank of Chinese Academy of Sciences (Shanghai, China). 293T and Lewis lung cancer (LLC) cells were cultured in Dulbecco`s modified Eagle`s medium supplemented with 10% fetal bovine serum (FBS) (HyClone, Logan, UT). H1299 and H1650 cells were cultured in RPMI1640 medium with 10% of FBS.

T cell mediated cytotoxicity and T cell migration assay

T cell-induced cytotoxicity was measured as described previously [12]. Briefly, NSCLC cells transfected with empty vector or miR-23a/27a/24 − 2 cluster expressing construct. After 48 hours of transfection, NSCLC cells were co-cultured with activated T cells (Chongqing Precision Biotech Co., Ltd, Chongqing, China) at a ratio of 1:20 for 6 hours, then cell death was measured by flow cytometry.

T cell migration assay was performed as described by Shang et al. [13]. Briefly, NSCLC cells were transfected with empty vector or miR-23a/27a/24 − 2 cluster overexpressing construct, and cell culture medium was changed after 48 hours of transfection. After 48 hours of cell culture with fresh medium, the supernatant was transferred into the lower chamber of transwell and 5Ⅹ10⁵ activated T cells in 100ul medium that have been incubated with anti-human CD8α APC-Cy7 antibody (BioLegend) added into the upper of transwell. After 2 hours co-incubation the migrated T cells were enumerated by flow cytometry.

Luciferase report assay

For miRNA reporter assay, miR-23a/27a/24 − 2 cluster expressing constructs or empty vectors were transfected into 293T cells that were transfected with a firefly luciferase reporter constructs containing the 3`-untranslational region (3` -UTR) of CBLB or MITF. For miR-23a/27a/24 − 2 cluster promoter luciferase assay, TCF4 expressing constructs or β-catenin expressing constructs or empty vectors were transfected into 293T cells that were transfected with a firefly luciferase reporter constructs containing miR-23a/27a/24 − 2 cluster promoter. The Renilla luciferase plasmid was cotransfected as a transfection control. After 72 hours of transfection, cells were harvested and subjected to measurement of luciferase activity.

Western blot, immunohistochemistry (IHC), immunofluorescence (IF), and coimmunoprecipitation (Co-IP) assay

Western blot, IHC, IF and Co-IP were performed as described as previously [14]. Primary antibodies against to eIF3B, MITF, MHC-Ⅰ, TCF4 and PD-L1 were purchased from Beijing Biosynthesis Biotechnology Co., Ltd (Beijing, China). β-catenin, Histone H3 and Actin antitodies were obtained from Cell Signaling Technology (Danvers, MA, USA). CD8 and CBLB antibodies were purchased from Abcam (Cambridge, MA, USA) and Proteintech (Wuhan, Hubei, China), respectively.

RNA analysis

Total RNAs were isolated using TRIzol reagent (Beyotime, Shanghai, China). mRNA sequencing and analysis was conducted by Shanghai Genechem Co., Ltd (Shanghai, China). miRNAs and gene mRNA expression levels are measured using qRT-PCR kit (Beyotime), and miRNA primers of has-miR-23a, has-miR-24-2 and has-miR-27a were purchased from GeneCopoeia (Rockville, MD, USA). miRNA in situ hybridization and score were performed as described by Nuovo [15] and Guo et al [16], respectively. Sequences of primmer and probes used in this study are summarized in Table S2.

Electrophoretic mobility shift assay (EMSA)

TCF4 binding site in miR-23a/27a/24 − 2 cluster was predicted using hTFtarget [17]. EMSA was performed using the LightShift chemiluminescence kit (Thermo Fisher Scientific (Waltham, MA, USA) according to manufacture`s instruction as described previously [14]. Sequences of probes are shown in Table S2.

Animal study

6-weeks old C57BL/6J female mice and SCID male mice (Beijing HFK Bioscience Co., Ltd, Beijing, China) were used for animal experiments. The effect of miR-23a/27a/24 − 2 cluster on lung cancer immune evasion was examined using C57BL/6J xenograft and SCID xenograft models. For generate C57BL/6J xenograft model, 1 × 10⁶ LLC cells that transfected with empty vector or miR-23a/27a/24 − 2 cluster plasmid or scramble or shRNA of miR-23a/27a/24 − 2 cluster in 100ul of phosphate-buffered saline (PBS) were transplanted subcutaneously on the backs of mice. For generate SCID xenograft model, 2 × 10⁶ H1299 cells that transfected with scramble plasmid or plasmid that expressing shRNA of miR-23a/27a/24 − 2 cluster in 100ul PBS were transplanted subcutaneously on the backs of mice. After 3days of tumor cell transplantation, mice were treated with or without 3× 10⁶ PBMC through the lateral tail vein injection. The tumor volumes were measured once a week.

The effects of miR-23a/27a/24 − 2 cluster on PD-1/PD-L1 blockade resistance was investigated using C57BL/6J xenograft models. 1 × 10⁶ LLC cells that transfected with empty vector or miR-23a/27a/24 − 2 cluster plasmid in 100ul of PBS were transplanted subcutaneously on the backs of mice. All group were treated with PD-L1 mAb (1mg/Kg body weight) every three days by intravenous (i.v.) injection.

To investigate the therapeutic effect of LF3 and PD-1/PD-L1 blockade combination on lung cancer with high expressing miR-23a/27a/24 − 2 cluster, C57BL/6J xenograft models were generated using miR-23a/27a/24 − 2 cluster high expressing LLC cells as described above. When tumor volumes are reached about 100mm³, mice were randomly divided into four groups and started to drug treatment. Control group mice were treated with IgG, and other groups were treated with PD-L1 mAb alone (1mg/Kg body weight, every three days) or LF3 alone (50mg/Kg body weight, every two days) or combination of PD-L1 mAb and LF3 by i.v. injection (Supplementary Fig. S1A).

To compare the effects of LF3 and 4EGI-1 treatment on PD-1/PD-L1 blockade therapeutic efficacy, C57BL/6J xenograft models were generated using miR-23a/27a/24 − 2 cluster high expressing LLC cells as described above. When tumor volumes are reached about 100mm³, mice were randomly divided into three groups and started to drug treatment. Control group mice were treated with PD-L1 mAb alone (1mg/Kg body weight, every three days), other groups were treated with PD-L1 mAb and LF3 or PD-L1 mAb and 4EGI-1 (75mg/Kg body weight, i.p. 5days a week) (Supplementary Fig. S1B). All animal experiment complied with the Army Medical University Policy on the Care and Use of Laboratory Animals.

Statistical analysis

Data were presented as the mean ± standard deviation of least three independent experiments. Statistical differences were analyzed by student`s T test or one-way analysis of variance using SAS statistical software. Survival curves were calculated using Kaplan-Meier survival analysis. Differences were considered statistically significant at a p value of less than 0.05.

High expression of miRNAs in miR-23a/27a/24 − 2 cluster closely associated with disease progression and immune evasion in NSCLC

To examine the correlation between miRNAs expression level of miR-23a/27a/24 − 2 cluster and NSCLC progression, patients with high expression of all miRNAs of miR-23a/27a/24 − 2 cluster in tumor tissues compared to adjacent tissues are classified into the high expression group. In contrast, patients with low or same expression level of all miRNAs of miR-23a/27a/24 − 2 cluster in tumor tissues compared to adjacent tissues are classified into the low expression group. Clinical data show that in NSCLC patients, miRNAs expression levels of miR-23a/27a/24 − 2 cluster are negatively correlated with overall survival rate (Fig. 1A) and disease-free survival rate (Fig. 1B). In addition, we found that group with high expression of miRNAs in miR-23a/27a/24 − 2 cluster had a lower number of infiltrating CD8⁺ T cells in tumor tissues (Fig. 1C). Further, Gene Ontology (GO) using mRNA sequencing data from miR-23a/27a/24 − 2 cluster silenced H1299 cells and control cells showed silencing of miR-23a/27a/24 − 2 cluster affects T cell activation, differentiation (Fig. 1D). Importantly, Gene Set Enrichment Analysis (GSEA) show miRNAs expression levels of miR-23a/27a/24 − 2 cluster negatively correlated with T cell mediated immunity in NSCLC cells (Fig. 1E). Consistent results were observed from the analysis of GEO dataset. As showing in Fig. 1F, GEO dataset analysis showed that miR-23a/27a/24 − 2 cluster expression level was associated with T cell activation and differentiation in NSCLC. In addition, target-based miRNA functional analysis [18] also showed that miRNAs of miR-23a/27a/24 − 2 cluster were associated with immune system process (Fig. 1G). Taken together, these findings suggesting that upregulated miRNAs of miR-23a/27a/24 − 2 cluster contribute to NSCLC progression by inhibiting T cell mediated immunity.

miRNAs of miR-23a/27a/24 − 2 cluster inhibit T cell mediated immune response in NSCLC

To investigate whether miRNAs of miR-23a/27a/24 − 2 cluster directly regulates T cell mediated immune response, T cells were co-cultured with miR-23a/27a/24 − 2 cluster overexpressing NSCLC cells. That transfection of miR-23a/27a/24 − 2 cluster expressing construct significantly upregulated all miRNAs of miR-23a/27a/24 − 2 cluster in NSCLC cells (Supplementary Fig. S2A). In vitro results show that overexpression of all miRNAs in miR-23a/27a/24 − 2 cluster significantly reduced T cell released IFN-γ (Fig. 2A), T cell-induced NSCLC cell death (Fig. 2B and Supplementary Fig. S3), and T cell migration (Fig. 2C). In addition, that overexpression of all miRNAs in miR-23a/27a/24 − 2 cluster (Supplementary Fig. S2B) dramatically promoted tumor growth (Fig. 2D), inhibited CD8⁺ T cells infiltrate into tumor tissues (Fig. 2E), and reduced IFN-γ expression (Fig. 2F) in the C57BL/6J xenograft models that constructed using LLC cells. In contrast, silencing of miR-23a/27a/24 − 2 cluster in LLC cells (Supplementary Fig. S2B) inhibited tumor growth (Fig. 2G), increased CD8⁺ T cells infiltration (Fig. 2H) and IFN-γ expression (Fig. 2I). These results were further confirmed using other xenograft models. Consistent with results from C57BL/6J xenograft models that silencing of miR-23a/27a/24 − 2 cluster in H1299 cells (Supplementary Fig. S2C) significantly inhibited tumor growth when the cells were inoculated in SCID mice that treated with or without peripheral blood mononuclear cells (PBMCs) (Fig. 2J-L). Notably, that PBMCs treatment more effectively increased the tumor inhibition rate of miR-23a/27a/24 − 2 cluster silencing in SCID mice models (Fig. 2M). Together, these data indicating a potential role of miR-23a/27a/24 − 2 cluster miRNAs in the inhibition of T cell mediated antitumor immune responses.

miRNAs of miR-23a/27a/24 − 2 cluster upregulated PD-L1 and downregulated MHC-Ⅰ expression in NSCLC

To investigate which proteins are involved in the inhibition of miRNAs in miR-23a/27a/24 − 2 cluster on T cell immune response, we performed proteomics analysis using NSCLC cells with high expression of all miRNAs in the miR-23a/27a/24 − 2 cluster and their control cells. As showing in Fig. 3A, many proteins expression was affected by overexpression of all miRNAs in the miR-23a/27a/24 − 2 cluster, including PD-L1 and MHC-Ⅰ, and that GO analysis showed that miR-23a/27a/24 − 2 cluster overexpression was correlated with MHC-Ⅰ mediated antigen processing and presentation pathway. Because PD-L1 and MHC-Ⅰ are key molecules for immune evasion and ICB resistance, we chose PD-L1 and MHC-Ⅰ proteins as targets for further experiments. Consistent with proteomics analysis, Western blot (Fig. 3B) and Flow cytometry analysis (Fig. 3C) showed the PD-L1 expression was upregulated by overexpression of miR-23a/27a/24 − 2 cluster miRNAs in NSCLC cells. The inhibitory effect of miR-23a/27a/24 − 2 cluster on MHC-Ⅰ expression in NSCLC cells also confirmed by Western blot (Fig. 3B) and immunofluorescence (IF) assay (Fig. 3D). In addition, immunohistochemistry (IHC) analysis of tumor tissues from a lung cancer model constructed using LLC cells in C57BL/6J mice showed that high expression of PD-L1 and low expression of MHC-Ⅰ in miR-23a/27a/24 − 2 cluster miRNAs high expressing group (Fig. 3E). These results have also been further confirmed in human NSCLC specimens by IHC analysis (Fig. 3F). Collectively, these data suggesting that miR-23a/27a/24 − 2 cluster may inhibit T cell mediated tumor immune response by upregulating PD-L1 and downregulating MHC-Ⅰ in NSCLC.

miRNAs of miR-23a/27a/24 − 2 cluster upregulated the expression of PD-L1 in NSCLC by targeting CBLB

Then, we investigated the regulatory mechanism of miR-23a/27a/24 − 2 cluster miRNAs on PD-L1 expression in NSCLC. From proteomics analysis, we found that CBLB was downregulated in group of miR-23a/27a/24 − 2 cluster overexpression (Fig. 3A), which is a negative regulator of PD-L1 in NSCLC [19]. The inhibitory effect of miRNAs of miR-23a/27a/24 − 2 cluster on CBLB expression in NSCLC cells at mRNA (Fig. 4A) and protein levels (Fig. 4B) was further confirmed by qRT-PCR and Western blot, respectively. Notably, our data show that overexpression of CBLB suppressed miRNAs of miR-23a/27a/24 − 2 cluster-induced upregulation of PD-L1 in NSCLC cells (Fig. 4C), suggesting that miRNAs of miR-23a/27a/24 − 2 cluster upregulate PD-L1 by inhibiting CBLB. In fact, we using computational algorithm analysis (targetscan.org) predicted the 3`-untranlated region (3`-UTR) of CBLB contains sequences that can bind with miR-23a and miR-27a (Fig. 4D). Importantly, a luciferase report assay showed that overexpression of miR-23a/27a/24 − 2 cluster significantly suppressed the luciferase level that regulated by wild 3`-UTR of CBLB, but, did not affect the luciferase expression level that regulated by mutant 3`-UTR of CBLB (Fig. 4E). The negative and positive correlation of miR-23a/27a/24 − 2 cluster with CBLB and PD-L1 expression was further confirmed by IHC analysis in tumor tissues that from C57BL/6J lung cancer models constructed using miR-23a/27a/24 − 2 cluster overexpressing LLC cells (Fig. 4F) and NSCLC patients (Fig. 4G). Taken together, these findings indicating that miRNAs of miR-23a/27a/24 − 2 cluster upregulate PD-L1 expression in NSCLC through inhibiting CBLB by directly targeting 3`-UTR of CBLB.

miRNAs of miR-23a/27a/24 − 2 cluster inhibits the expression of MHC-Ⅰ in NSCLC by targeting MITF

We also investigated the regulatory mechanism of miR-23a/27a/24 − 2 cluster on MHC-Ⅰ expression. From proteomic results, we found that eIF3B was upregulated in miR-23a/27a/24 − 2 cluster overexpressed groups (Fig. 3A). In addition, computational algorithm analysis showed that 3`-UTR of MITF contains sequences that can bind with all miRNAs of miR-23a/27a/24 − 2 cluster (Fig. 5A). According to Santasusagna et al. report, that MITF inhibits the expression of eIF3B, while eIF3B inhibits the expression of MHC-Ⅰ [20], suggesting that miRNAs of miR-23a/27a/24 − 2 cluster in NSCLC may downregulate MHC-Ⅰ expression through upregulating eIF3B by targeting MITF. As expected, in vitro experiment results showed that overexpression of miR-23a/27a/24 − 2 cluster dramatically downregulated the expression of MITF and MHC-Ⅰ expression in NSCLC cells, while, upregulated eIF3B expression (Fig. 5B). Also, downregulated expression of MITF by miR-23/27a/24 − 2 cluster at mRNA level was demonstrated in NSCLC cells (Fig. 5C). Importantly, that overexpression of MITF or silencing eIF3B restored MHC-Ⅰ expression that inhibited by miR-23a/27a/24 − 2 cluster (Fig. 5D). The miR-23a/27a/24 − 2 cluster-induced upregulation of eIF3B was also inhibited by overexpression of MITF (Fig. 5D). Further, we using luciferase reporter assay investigated whether miRNAs in miR-23a/27a/24 − 2 cluster inhibit MITF expression by targeting 3`-UTR of MITF. As showing in Fig. 5E, overexpression of miR-23a/27a/24 − 2 cluster significantly suppressed the luciferase level that regulated by wild 3`-UTR of MITF, but, did not affect the luciferase expression level that regulated by mutant 3`-UTR of MITF. Finally, the correlation among the expression levels of miR-23a/27a/24 − 2 cluster, MITF, eIF3B and MHC-Ⅰ was further confirmed by IHC analysis of tumors from C57BL/6J xenograft models that constructed using miR-23a/27a/24 − 2 cluster overexpressing LLC cells (Fig. 5F) and patients with NSCLC (Fig. 5G). Collectively, these findings suggesting that miRNAs of miR-23a/27a/24 − 2 cluster inhibits MHC-Ⅰ expression in NSCLC through upregulating eIF3B by directly targeting MITF.

miRNAs of miR-23a/27a/24 − 2 cluster maintain its expression through β-catenin/TCF4 axis in NSCLC

To investigate the regulatory mechanism of miR-23a/27a/24 − 2 cluster miRNAs in NSCLC, we have screened candidate transcription factors that may be involved in the regulation of miR-23a/27a/24 − 2 cluster expression, and found that TCF4 can bind with promoter of miR-23a/27a/24 − 2 cluster (Fig. 6A). TCF4 is an effector of Wnt/β-catenin signaling and involved in miRNAs expression by binding to promoter region of miRNAs [21]. In addition, our previous study has shown that miRNAs of miR-23a/27a/24 − 2 cluster stimulate TCF/LEF complex regulated gene expression by activating Wnt/β-catenin signaling in NSCLC [9]. Thus, we proposed that miR-23a/27a/24 − 2 cluster maintenance its expression through β-catenin/TCF4 axis. To prove this hypothesis, we first confirmed that TCF4 whether regulates miR-23a/27a/24 − 2 cluster expression. Our results show that overexpression of TCF4 significantly upregulated expression of miR-23a, miR-27a and miR-24-2 (Fig. 6B) and luciferase transcription that regulated by miR-23a/27a/24 − 2 cluster promoter (Fig. 6C). In addition, EMSA results show that TCF4 directly interact with miR-23a/27a/24 − 2 cluster promoter site that containing TCF4 binding sequences, while, mutation of binding sequence significantly reduced the interaction between TCF4 and miR-23a/27a/24 − 2 cluster promoter (Fig. 6D). Collectively, these results indicating that TCF4 promotes miRNAs expression of miR-23a/27a/24 − 2 cluster by interacting to miR-23a/27a/24 − 2 cluster promoter. Then, we investigated that β-catenin whether promotes TCF4 mediated expression of miR-23a/27a/24 − 2 cluster miRNAs. The EMSA results show that overexpression of β-catenin increased the interaction between TCF4 and miR-23a/27a/24 − 2 cluster promoter (Fig. 6E), miR-23a/27a/24 − 2 cluster promoter regulated luciferase transcription (Fig. 6F), and miRNAs expression of miR-23a/27a/24 − 2 cluster (Fig. 6G). Notably, inhibition of the interaction between β-catenin and TCF4 by small molecule LF3 dramatically inhibited β-catenin overexpression-induced upregulation of miR-23a/27a/24 − 2 cluster miRNAs (Fig. 6H). Together, these findings indicating that miR-23a/27a/24 − 2 cluster maintains its expression through β-catenin/TCF4 axis in NSCLC.

Targeting eIF3B pathway dramatically enhanced therapeutic efficacy of PD-1/PD-L1 blockade in lung cancers with high expression of miRNAs in miR-23a/27a/24 − 2 cluster

Given that miRNAs of miR-23a/27a/24 − 2 cluster modulate the expression of key molecules associated with PD-1/PD-L1 blockade therapy efficacy, we investigated whether high expression of miRNAs in miR-23a/27a/24 − 2 cluster leads to resistance to PD-1/PD-L1 blockade. As expected that overexpression of miR-23/27a/24 − 2 cluster miRNAs reduced the sensitivity of tumors to PD-L1 mAb treatment (Fig. 7A) and suppressed the infiltration of CD8⁺ T cells into tumors in C57BL/6J mice models that constructed using LLC cells (Fig. 7B).

Further, given that miR-23a/27a/24 − 2 cluster maintains its expression through β-catenin/TCF4 axis and downregulates MHC-Ⅰ expression through eIF3B pathway, we investigated whether these axes could be pharmacologically targeted to enhance therapeutic efficacy of PD-1/PD-L1 blockade. Animal experiment results show that blockade of the interaction between β-catenin and TCF4 by LF3 (Fig. 7C) significantly suppressed growth of tumors with high expression of miR-23a/27a/24 − 2 cluster miRNAs compared to control group (Fig. 7D-F). Notably, compared to single drug treatment, combination of LF3 and mAb of PD-L1 more significantly inhibited tumor growth in miR-23a/27a/24 − 2 cluster high expressing lung cancers (Figs. 7D-F). In addition, LF3 treatment suppressed miRNAs expression of miR-23a/27a/24 − 2 cluster (Fig. 7G), PD-L1 expression, and upregulated MHC-Ⅰ expression (Fig. 7H). Notably, that combination of PD-L1 mAb and LF3 more significantly increased infiltration of CD8⁺ T cells into tumor tissues (Fig. 7I). Then, we compared the promoting effects of targeting the β-catenin/TCF4 axis and targeting the eIF3B pathway on PD-1/PD-L1 blockade therapy. Because, small molecule 4EGI-1 impedes eIF3B binding to the translation initiation assembly and inhibits MITF/eIF3B pathway [20, 22], we used 4EGI-1 as inhibitor of eIF3B pathway. The animal experiment results show that targeting of eIF3B pathway by 4EGI-1 more significantly enhanced PD-L1 mAb therapeutic efficacy in miR-23a/27a/24 − 2 cluster high expressing lung cancer than inhibiting β-catenin and TCF4 interaction by LF3 treatment (Fig. 7J). In addition, we demonstrated that combination of 4EGI-1 and PD-L1 mAb more significantly increased CD8⁺ T cells infiltrate into tumor tissues (Fig. 7K). We also demonstrated that 4EGI-1 treatment caused upregulation of MHC-Ⅰ expression, but did not suppress miR-23a/27a/24 − 2 cluster-induced high expression of PD-L1 (Fig. 7L). Taken together, these data indicating that targeting eIF3B can significantly enhance the therapeutic efficacy of PD-1/PD-L1 blockade in miR-23a/27a/24 − 2 cluster overexpressing lung cancer.

In this study, we investigated the molecular mechanism that miRNAs of miR-23a/27a/24 − 2 cluster led to immune evasion and PD-1/PD-L1 blockade resistance in NSCLC. Growing evidences has shown that PD-L1 [23] and MHC-Ⅰ [24] are key molecules associated with tumor immune response. PD-L1 is an immune checkpoint protein that express on the surface of tumor cells and inactivate T cells by bindings to PD-1 on the T cell surface. Thus, abnormally high expression of PD-L1 is an important mechanism for cancer immune evasion. In addition, downregulation of MHC-Ⅰ also is one of the important tumor immune evasion mechanisms. MHC-Ⅰ loads peptides and presents them on the surface of cells, which provided the immune system with the signal to detect and eliminate abnormal cells [25]. When the peptides loaded by MHC-Ⅰ are tumor-associated antigen, the CD8⁺ T cells would be activated following the antigen recognition and then induce the immune killing to cancer cells [25]. Unfortunately, low expression or loss of MHC-Ⅰ is a frequent event in various cancers, including lung cancer [26, 27], which leading to T cells doesn`t detect cancer cells, allowing cancer cells to survive [27]. In this study, we demonstrated that overexpression of miRNAs in miR-23a/27a/24 − 2 cluster promotes NSCLC immune evasion and PD-1/PD-L1 blockade resistance. Our data show that miR-23a/27a/24 − 2 cluster upregulated PD-L1 expression in NSCLC by directly targeting the PD-L1 negative regulator CBLB, and downregulated MHC-Ⅰ expression through upregulating MHC-Ⅰ negative regulator eIF3B by directly targeting MITF. The promoting effect of miRNAs of miR-23a/27a/24 − 2 cluster on tumor immune evasion also reported in other tumors. A report showed that miR-27a inhibits MHC-Ⅰ expression and CD8⁺ T cell infiltration in colorectal cancer [24]. Lin et al. reported that miR-23a upregulated in CD8⁺ cytotoxic T lymphocytes and targeting miR-23a in CD8⁺ cytotoxic T lymphocytes prevents tumor-dependent immunosuppression in lung cancer [28]. In addition, studies show that upregulated expression of miR-23a and miR-27a in macrophages contribute to cancer immune evasion by upregulating PD-L1 expression through PTEN/PIK3 pathway in liver cancer [29] and breast cancer [30], respectively. Taken together, these findings indicating that upregulated expression of miRNAs in miR-23a/27a/24 − 2 cluster is a promoting factor for tumor immune evasion, and plays its role by upregulating PD-L1 and downregulating MHC-Ⅰ. Our study provided a novel mechanism of upregulating PD-L1 and downregulating MHC-Ⅰ expression in NSCLC.

In this study, we further elucidated the molecular mechanism by which miRNAs of miR-23a/27a/24 − 2 maintain high expression in NSCLC. Abnormally upregulated expression of miRNAs in miR-23a/27a/24 − 2 cluster was identified in about 50% cases of early-stage NSCLC, and closely correlated with disease progression [9], but, the mechanism of miR-23a/27a/24 − 2 cluster high expression is not clear. In this study, we demonstrated that TCF4 increases miR-23a/27a/24 − 2 cluster expression by directly binding to its promoter region, and this interaction was promoted by β-catenin. Importantly, our previous study has shown that miRNAs of miR-23a/27a/24 − 2 cluster promotes TCF/LEF complex mediated gene expression through activating Wnt/ β-catenin signaling by targeting negative regulators of Wnt/ β-catenin signaling in NSCLC [9]. Collectively, these findings suggesting that miR-23a/27a/24 − 2 cluster maintain its expression levels by activating β-catenin/TCF4 axis in NSCLC.

Finally, we proposed a novel strategy for treating NSCLC with high expression of miR-23a/27a/24 − 2 cluster. Our studies show that miR-23a/27a/24 − 2 cluster activates β-catenin signaling [9] and eIF3B pathway, and these both pathways are contribute to PD-1/PD-L1 blockade resistance [20, 31]. In this study, we demonstrated that both targeting β-catenin/TCF4 axis or eIF3B pathway can significantly enhance PD-L1 mAb therapeutic efficacy. However, targeting eIF3B pathway more effectively enhanced PD-L1 mAb therapeutic efficacy than targeting β-catenin/TCF4 axis. These results may be caused by the differential regulation of PD-L1 and MHC-Ⅰ expression by β-catenin/TCF4 and eIF3B pathway. Clinical studies have shown that the success of PD-1/PD-L1 blockade is closely correlated with the expression level of PD-L1 in tumor cells [32, 33]. Zhang et al. also reported that upregulation of PD-L1 expression can enhance PD-1/PD-L1 blockade therapeutic effects [34]. Our data show that targeting β-catenin/TCF4 axis upregulated MHC-Ⅰ expression, but decreased the expression of PD-L1. In contrast, targeting eIF3B pathway did not inhibit miR-23a/27a/24 − 2 cluster-induced high expression of PD-L1, while, significantly upregulated MHC-Ⅰ expression levels.

In summary, miRNAs of miR-23a/27a/24 − 2 cluster maintain their expression through activating β-catenin/TCF4 axis in NSCLC. High expressed miRNAs of miR-23a/27a/24 − 2 cluster upregulated PD-L1 expression by targeting PD-L1 negative regulator CBLB, and downregulated MHC-Ⅰ expression through upregulating eIF3B expression by targeting MITF (Fig. 8 left). In addition, this study provides a therapeutic strategy that targeting eIF3B pathway can significantly enhance PD-1/PD-L1 blockade therapeutic efficacy in lung cancer with high expressing miR-23a/27a/24 − 2 cluster miRNAs through restoring MHC-Ⅰ expression and keep miR-23a/27a/24 − 2 cluster-induced high expression of PD-L1 (Fig. 8 right).

Ethical approval

This study was approved by the Ethics Committee of Daping Hospital of Army Medical University (Medical Research Ethics Review No.120), and all patients obtained informed consent before enrollment. All mouse procedures were approved by the Laboratory Animal Welfare and Ethics Committee of Army Medical University (AMUWEC20211547).

Competing interests

The authors declare no competing interests.

Funding

This work was supported by the National Natural Science Foundation of China (81672283, to H.J.), the Chongqing Natural Science Foundation (cstc2021jcyj-msxmX0350, to H.J.), and the Beijing Natural Science Foundation (7232168, to R.-T.W.).

Author Contribution

H.J and C.X.X designed experiments and prepared the manuscript. H.J, C.X.X, H.L, X.R.G , J.C and Q.L performed in vitro and in vivo data analysis. J.C performed bioinformatics analysis and draw a graphic abstract. H.L, Q.L, J.C, and X.F performed in vitro and animal experiments. W.Z, R.T.W, X.D.H, and W.G participated in human sample collection and clinical data analysis.

Data Availability

Data is provided within the manuscript or supplementary information files.

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The miR-23a/27a/24-2 cluster drives immune evasion and resistance to PD- 1/PD-L1 blockade in non-small cell lung cancer

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Abstract

Figures

Introduction

Methods

Human specimens and Cell culture

T cell mediated cytotoxicity and T cell migration assay

Luciferase report assay

Western blot, immunohistochemistry (IHC), immunofluorescence (IF), and coimmunoprecipitation (Co-IP) assay

RNA analysis

Electrophoretic mobility shift assay (EMSA)

Animal study

Statistical analysis

Results

miRNAs of miR-23a/27a/24 − 2 cluster inhibit T cell mediated immune response in NSCLC

miRNAs of miR-23a/27a/24 − 2 cluster upregulated PD-L1 and downregulated MHC-Ⅰ expression in NSCLC

miRNAs of miR-23a/27a/24 − 2 cluster upregulated the expression of PD-L1 in NSCLC by targeting CBLB

miRNAs of miR-23a/27a/24 − 2 cluster inhibits the expression of MHC-Ⅰ in NSCLC by targeting MITF

miRNAs of miR-23a/27a/24 − 2 cluster maintain its expression through β-catenin/TCF4 axis in NSCLC

Discussion

Conclusion

Declarations

Funding

Author Contribution

Data Availability

References

Additional Declarations

Supplementary Files

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