The innovative checkpoint inhibitors of lung adenocarcinoma, cg09897064 methylation and ZBP1 expression reduction, have implications for macrophage polarization and tumor growth in lung cancer

Wang, Ailing; Zheng, Wei-sha; Luo, Zhen; Bai, Lian; Zhang, Shi

doi:10.1186/s12967-024-04995-1

The innovative checkpoint inhibitors of lung adenocarcinoma, cg09897064 methylation and ZBP1 expression reduction, have implications for macrophage polarization and tumor growth in lung cancer

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Published: 18 February 2024

Volume 22, article number 173, (2024)
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The innovative checkpoint inhibitors of lung adenocarcinoma, cg09897064 methylation and ZBP1 expression reduction, have implications for macrophage polarization and tumor growth in lung cancer

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Ailing Wang³,
Wei-sha Zheng³,
Zhen Luo^1,2,3,
Lian Bai³ &
…
Shi Zhang ORCID: orcid.org/0000-0002-5972-6733^1,2

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Abstract

Lung cancer, a prevalent and aggressive disease, is characterized by recurrence and drug resistance. It is essential to comprehend the fundamental processes and discover novel therapeutic objectives for augmenting treatment results. Based on our research findings, we have identified a correlation between methylation of cg09897064 and decreased expression of ZBP1, indicating a link to unfavorable prognosis in patients with lung cancer. Furthermore, these factors play a role in macrophage polarization, with ZBP1 upregulated in M1 macrophages compared to both M0 and M2 polarized macrophages. We observed cg09897064 methylation in M2 polarization, but not in M0 and M1 polarized macrophages. ATACseq analysis revealed closed chromatin accessibility of ZBP1 in M0 polarized macrophages, while open accessibility was observed in both M1 and M2 polarized macrophages. Our findings suggest that ZBP1 is downregulated in M0 polarized macrophages due to closed chromatin accessibility and downregulated in M2 polarized macrophages due to cg09897064 methylation. Further investigations manipulating cg09897064 methylation and ZBP1 expression through overexpression plasmids and shRNAs provided evidence for their role in modulating macrophage polarization and tumor growth. ZBP1 inhibits M2 polarization and suppresses tumor growth, while cg09897064 methylation promotes M2 polarization and macrophage-induced tumor growth. In mechanism investigations, we found that cg09897064 methylation impairs CEBPA binding to the ZBP1 promoter, leading to decreased ZBP1 expression. Clinical experiments were conducted to validate the correlation between methylation at cg09897064, ZBP1 expression, and macrophage M2 polarization. Targeting these factors may hold promise as a strategy for develo** innovative checkpoint inhibitors in lung cancer treatment.

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Introduction

The Global Cancer Report reveals that lung cancer is a widespread malignant tumor, with a mortality rate of 82–89% worldwide. In China, 730,000 new cases and 610,000 deaths are reported annually, with a 5-year survival rate of 11–17%- a grave health hazard and social burden [1,2,3].Of all the lung cancer types, adenocarcinoma is the most prevalent, accounting for more than 50%of cases [4, 5].

In the past decade, lung cancer treatment has seen advances in early detection, targeted therapy, and immunotherapy, which have extended patient survival. However, drug resistance and recurrence remain issues [5]. Consequently, it is essential to investigate multiple treatment options in order to tackle these problems and progress lung cancer treatment.

The development of lung cancer is profoundly impacted by epigenetic alterations, both genetically and environmental. Epigenetic factors, such as abnormal DNA methylation induced by risk factors like smoking and chronic lung disease, can deactivate tumor suppressor genes and activate oncogenes. This process ultimately leads to the development of lung cancer. Furthermore, site-specific methylation may contribute to treatment resistance, making epigenetic changes promising therapeutic targets for lung cancer [6,7,8].

The understanding of tumor immune infiltration's molecular mechanisms has led to the development of multiple immune checkpoints and targeted drugs. This progress has made immunotherapy a significant treatment modality for lung cancer. Monoclonal antibodies targeting PD-1/PD-L1 have been extensively utilized in cancer treatment [9, 10]. However, challenges such as drug resistance, recurrence, and tumor heterogeneity still hinder the efficacy of immunotherapy. Further investigation is necessary to expand our understanding of immune infiltration processes in lung cancer. This will help in the development of new immune checkpoints and complementary immune-targeted therapies, ultimately improving treatment outcomes and patients' quality of life. Epigenetic regulation may possibly augment immune infiltration [9, 10]. Currently, immune therapy primarily targets downstream immune co-stimulatory protein molecules. By regulating epigenetics upstream and blocking immune checkpoint transcription, immune infiltration can be fundamentally improved. Epigenetic drugs [11, 12] primarily target DNA methylation, the most prevalent epigenetic alteration.

This study examines the mechanistic association between cg09897064 methylation on Z-DNA binding protein 1 (ZBP1) in lung adenocarcinoma. It explores the implications for patients' prognosis, macrophage polarization, and tumor growth. Our results demonstrate that cg09897064 methylation leads to a decrease in ZBP1 expression. This, in turn, causes macrophages to polarize towards the M2 phenotype and promotes tumor growth. Mechanistically, we discovered that cg09897064 methylation impairs CCAAT Enhancer Binding Protein Alpha (CEBPA) binding to the ZBP1 promoter, resulting in decreased ZBP1 expression. Clinical experiments were conducted to validate the correlation between methylation at cg09897064, ZBP1 expression, and macrophage M2 polarization. The potential for the invention of pioneering checkpoint inhibitors in lung cancer treatment is great if these factors are targeted.

Materials and methods

High-throughput data analysis

Data of lung adenocarcinoma and its neighboring normal tissues' transcript some and DNA methylation were procured from The Cancer Genome Atlas(TCGA)database. In the TCGA database, a comprehensive search was conducted for all expression microarray datasets associated with lung adenocarcinoma up until January 2022. Animal and cell model data related to lung adenocarcinoma were excluded. The inclusion criteria were as follows: (1) patients diagnosed with lung adenocarcinoma, (2) availability of both transcriptomic and DNA methylation data, (3) presence of patient survival information, and (4) availability of downloadable data for further secondary analysis.

To guarantee the comparability of the cohorts, we used the sva R package and Perl's ComBat normalization technique to co-normalize the data into a unified cohort. The raw data was re-normalized,with the batch correction of DNA methylation microarray data done through the minfi,impute,and wateRmelon R packages [Full size image

ZBP1 is known to have 4 methylation sites: cg07912689, cg09897064, cg17306740, and cg24380059. The correlation analysis (Fig. 1C-F) suggests that cg09897064 methylation has the highest correlation with ZBP1 mRNA expression (highest absolute correlation coefficient of -0.426, lowest P-value). Hence, we infer that the methylation of cg09897064 greatly impacts the transcription of ZBP1 mRNA.

Association between cg09897064 methylation on ZBP1 and lung adenocarcinoma prognosis and macrophage polarization

Further analysis of lung adenocarcinoma data from TCGA database revealed the following findings: a comparison of cg09897064 methylation levels in various tissue samples revealed that cg09897064 had a greater methylation in adjacent normal tissue than lung adenocarcinoma tissue (P < 0.001). Additionally, lung adenocarcinoma tissue from deceased patients had higher methylation levels of cg09897064 compared to surviving patients (P < 0.001), shown in Fig. 2A. The results indicate that the presence of cg09897064 methylation is linked to an unfavorable prognosis among individuals with lung adenocarcinoma.

The expression of ZBP1 mRNA was also examined in different tissue samples. A marked difference in ZBP1 mRNA expression betweenlung adenocarcinoma tissue and its neighboring normal tissue was noted (P < 0.001). Furthermore, ZBP1 mRNA expression was significantly higher in lung adenocarcinoma tissue from surviving patients compared to deceased patients(P < 0.01),shown in Fig. 2B.The expression of ZBP1 mRNA being low appears to be a risk factor for a dismal outcome in lung adenocarcinoma patients, as these results demonstrate.

Patients were categorized into two groups, namely the "high methylation of cg09897064 and low expression of ZBP1 mRNA group" and the "low methylation of cg09897064 and high expression of ZBP1 mRNA group", based on the median values of cg09897064 methylation level and ZBP1 mRNA expression. Survival analysis of these two groups revealed that patients in the "low methylation of cg09897064& high expression of ZBP1 mRNA group" had a significantly better prognosis compared to those in the "high methylation of cg09897064& low expression of ZBP1 mRNA group" (P < 0.001), shown in Fig. 2C.

Furthermore, the impact of cg09897064 methylation on the tumor immune microenvironment was investigated using the "CIBERSORT" algorithm. A comparison of the two groups of lung adenocarcinoma tissue revealed considerable disparities in the proportions of different immune cells (P < 0.01). Particularly, the "low methylation of cg09897064& high expression of ZBP1 mRNA group" showed a marked rise in M1 macrophages and a noteworthy decrease in M2 macrophages when compared to the "high methylation of cg09897064& low expression of ZBP1 mRNA group", as depicted in Fig. 2D.

To sum up, the evidence implies that cg09897064 methylation is linked to a lack of ZBP1 expression and encourages the polarization of M2 macrophages. In addition, the methylation of cg09897064 is associated with a negative prognosis in patients with lung adenocarcinoma.

In vitro induction of ZBP1 down-regulation and macrophage M2 polarization by cg09897064 methylation

To explore the association between cg09897064 methylation and the manifestation of ZBP1 during macrophage polarization, a series of experiments were conducted. CD14⁺ mononuclear macrophages were exposed to IFNγ and IL4, respectively, to provoke M1 and M2 polarization. The CD14⁺ mononuclear macrophages were sourced from Pricella Life Science&Technology Company in Wuhan, China, and confirmed through immunofluorescence staining (Additional file 1: Figure S1).

A successful establishment of an in vitro model for M1 and M2 polarized macrophages demonstrated a significant increase in ZBP1 expression in M1 macrophages, as compared to both M0 and M2 polarized macrophages, as depicted in Fig. 3A-D through Western blot analysis.

To further investigate the link between cg09897064 methylation and ZBP1 expression, we conducted MSP and BSAS. Our research uncovered cg09897064 methylation in M2 macrophage polarization, yet not in M0 and M1 polarized macrophages. This observation may provide an explanation for the decreased expression of ZBP1 in M2 polarized macrophages, shown in Fig. 3E-F.

In the subsequent investigation, we focused on the downregulation of ZBP1 in M0 polarized macrophages. The ATACseq analysis showed that the chromatin accessibility of ZBP1 and its promoter region was closed in M0 polarized macrophages, but open in both M1 and M2 polarized macrophages. This finding suggests that the downregulation of ZBP1 in M0 macrophages may be associated with the differential chromatin accessibility dynamics during macrophage polarization, shown in Fig. 3G.

To investigate the impact of cg09897064 methylation and ZBP1 expression on macrophage polarization from M1 to M2,we constructed ZBP1-unmethylation overexpression plasmid (ZBP1-U), ZBP1-methylation overexpression plasmid(cg09897064 methylation) (ZBP1-M), and a blank control plasmid(Ctrl), which were then transfected into mononuclear macrophages. Flow cytometry, ELISA, and Western blotting were performed to evaluate the expression of M1 polarized markers(iNOS,CD86,and IL1) and M2 polarized markers(ARG1,CD206,IL10). Our findings demonstrated that the expression of M1 polarized markers(iNOS,CD86,and IL1)significantly decreased following ZBP1-U transfection,when compared to ZBP1-M or Ctrl plasmids (P < 0.001). In Fig. 4,it can be seen that the expression of M2 polarized markers(ARG1,CD206,IL10) was noticeably reduced when comparing macrophage polarization from M1 to M2 after ZBP1-U transfection to cg09897064 methylation on ZBP1(P < 0.001).

These findings suggest that ZBP1 inhibits macrophage polarization from M1 to M2, and that cg09897064 methylation suppresses ZBP1 expression, promoting macrophage M2 polarization.

In vivo regulation of macrophage M2 polarization and mononuclear macrophages-induced tumor growth by cg09897064 methylation

An in vivo experiment was conducted to explore the influence of cg09897064 methylation and ZBP1 expression on the interactions between macrophage and tumor cells, particularly in terms of the promotion of tumorigenesis by M2 macrophages. CD14⁺ mononuclear macrophages were infected with ZBP1-U, ZBP1-M, shZBP1, or control lentivirus and mixed with A549 lung carcinoma epithelial cells. Mice had their armpits implanted with the cell mixture subcutaneously for a period of 6 weeks. Lentiviral transduction enabled stable knockdown of ZBP1 in the shZBP1 group, compared to the control virus-transduced cells. Furthermore, the ZBP1-U plasmid significantly upregulated ZBP1 expression compared to the ZBP1-M transduced cells (P < 0.001) (Fig. 5A and B).Visible distinctions in the proliferation of primary tumors after subcutaneous injection of the cell mixture were seen between the control group and the shZBP1 group(Figs. 5C and D),with no noteworthy variations in the mice's body weight(Additional file 1: Figure S2 and Figs. 5E).

Analysis of the expression of CD206 was performed using immunohistochemistry(IHC), as demonstrated in Fig. 5F. Results showed that the ZBP1-U expression group had a lower CD206 expression in the tumor region compared to the ZBP1-M expression group, while the shZBP1 group had a higher CD206 expression in the tumor region. This assessment was done to evaluate the possible roles of cg09897064 methylation and ZBP1 expression in macrophage M2 polarization-induced tumor growth. Quantitative analysis using the log IOD in Image-Pro Plus 6.0 further confirmed the correlation between CD206 expression levels and tumor growth (Fig. 5G).

Impaired CEBPA binding to ZBP1 promoter domain due to cg09897064 methylation

We conducted an analysis of the CpG island methylation at the cg09897064 site on ZBP1 using the National Center forBiotechnology Information (NCBI) database in order to understand the mechanism by which cg09897064 hampers ZBP1 expression. We found that the methylated CpG islands of cg09897064 were located upstream of the ZBP1 transcript's TSS at -1100 bp and -1096 bp, within the promoter domain of ZBP1.We hypothesized that methylation of cg09897064 would impede the promoter's transcriptional activity. To verify this, luciferase reporter plasmids were constructed with either unmethylated (U) or methylated (M) regions (Fig. 6A). These plasmids were transfected into CD14 + mononuclear macrophages to assess their transcriptional activity.The pGL3-Promoter-U plasmid, in comparison to the control group and the methylated promoter regions, exhibited a marked rise in luciferase activity (P < 0.001), as demonstrated in Fig. 6A.

To further provide solid evidence for this mechanism, we identified CEBPA as the transcription factor (TF) that binds to the CpG island regions through motif analysis of our previous ATACseq data (Fig. 6B). EMSA confirmed that the biotin-labeled promoter-U has the ability to bind to CEBPA, while the promoter-M does not. This is illustrated in Fig. 6C.The methylation of CpG islands in cg09897064 seems to hinder the transcriptional activity of the promoter and also inhibit the binding of CEBPA to the ZBP1 promoter. This conclusion is supported by the findings presented in Fig. 6D.

Clinical validation of the correlation between methylation at cg09897064, ZBP1 expression, and macrophage M2 polarization

A total of 21 patients with lung adenocarcinoma were included, and the basic information of the patients is shown in Additional file 1: Table S4. Methylation-specific PCR (MSP) revealed the presence of cg09897064 site methylation in 47.6% (10/21) of the adenocarcinoma patients' pathological tissues. Further Western blot analysis showed significantly reduced expression of ZBP1 protein in patients with methylation at the cg09897064 site, P < 0.001 (Fig. 7). Flow cytometry analysis indicated a significant increase in the proportion of M2 macrophages in the adenocarcinoma tissues of patients with cg09897064 site methylation compared to non-methylated patients, P < 0.001 (Fig. 7).

Discussion

Despite significant progress in treatment, lung cancer is still a highly unfavorable disease, with recurrent recurrence and drug resistance. Therefore, it is essential to expand our knowledge of the root cause of lung cancer and discover new therapeutic targets. Adenocarcinoma is highly prevalent in clinical settings, establishing it as the most common malignant tumor and one of the most widespread. Our research has unveiled that cg09897064 methylation and reduced expression of ZBP1 serve as adverse prognostic factors in lung cancer patients. The results of subsequent experiments have been convincing, demonstrating the mechanistic part they play in the transformation of macrophages from an M1 to M2 phenotype, thus promoting tumor progression through M2 macrophages. These discoveries present promising prospects for the creation of novel checkpoint inhibitors targeting lung cancer.

Evidence has been steadily increasing that macrophage polarization is a major factor in the commencement and advancement of tumors [18,19,20]. Macrophages are a highly plastic cell population that can polarize into distinct functional states depending on the tumor background and disease stage. It is important to recognize that various factors, including cellular and non-cellular components of the immune microenvironment, can influence macrophage polarization in the tumor context.

Immune cells and non-cellular factors within the tumor microenvironment play a critical role in regulating the polarization state of macrophages. Immune cells such as lymphocytes and dendritic cells can induce specific polarization states in macrophages through the secretion of cytokines and stimulation molecules. Furthermore, tumor cells and non-cellular components, such as tumor-secreted cytokines, growth factors, and tumor-associated matrix components, can directly or indirectly impact macrophage polarization. Modulation of these factors can lead to changes in macrophage phenotype and function, thereby influencing tumor growth, invasion, and treatment response.

The polarization state of macrophages is crucial for tumor development and therapeutic responses. In early disease stages, an increase in M1 macrophages can inhibit tumor growth through the release of inflammatory mediators and pro-inflammatory cytokines. However, in late-stage disease, the tumor microenvironment can promote the increase of M2 macrophages, which exhibit immune-suppressive activity and contribute to tumor progression, angiogenesis, and metastasis. Therefore, modulating the polarization state of macrophages may be an important strategy for cancer therapy [21,22,23,24].

ZBP1 is a protein which identifies and interacts with Z-DNA, a particular structure of DNA. It regulates gene expression, repairs DNA damage, and contributes to immune response against viral infections.ZBP1 has significant involvement in multiple cellular processes, and its therapeutic potential may be uncovered through additional research [25,26,27,28].Our study enhances current understanding, offering new insights into the mechanisms behind macrophage polarization and its impact on tumor growth.In particular,we have determined that ZBP1 plays a crucial role in controlling M1 macrophage polarization. ZBP1, also known as Z-DNA binding protein 1, has been found to promote M1 polarization by activating pro-inflammatory signaling pathways. Through a series of in vitro and in vivo experiments, we have demonstrated that ZBP1 overexpression enhances M1 polarization and subsequently suppresses tumor growth.

Furthermore, our study has revealed a novel epigenetic mechanism involving cg09897064, which acts as a negative regulator of ZBP1 expression. We have observed that cg09897064 methylation leads to the downregulation of ZBP1, consequently promoting M2 polarization of macrophages. The increased tumor growth and progression are linked to the transition to an M2 phenotype. Our findings ultimately enhance the understanding of the complex association between macrophage polarization and tumor growth. By elucidating the molecular mechanisms involved, particularly the role of ZBP1 and cg09897064, our research provides potential targets for therapeutic interventions aimed at modulating macrophage polarization and inhibiting tumor growth.

Furthermore, we conducted clinical validation based on pathological specimens from 21 patients with lung adenocarcinoma and found that cg09897064 methylation is associated with ZBP1 expression and macrophage polarization, representing a novel immune checkpoint. Future studies with larger sample sizes are needed to further confirm the clinical value of cg09897064 methylation as an immune checkpoint in lung adenocarcinoma.

Conclusion

Our research has identified cg09897064 methylation and reduced expression of ZBP1 as adverse prognostic factors in lung cancer patients. Specifically, we have discovered that ZBP1 promotes M1 polarization, while cg09897064 inhibits ZBP1 expression, leading to M2 polarization and tumor progression. These discoveries offer promising possibilities for the creation of groundbreaking checkpoint inhibitors for the treatment of lung cancer.

Availability of data and materials

The TCGA and GEO database contain all the data produced and/or scrutinized during this research. The study's series in GEO was identified as GSE245440.

Abbreviations

ZBP1:: Z-DNA binding protein 1
CEBPA:: CCAAT Enhancer Binding Protein Alpha
TCGA:: The Cancer Genome Atlas database
ATACseq:: Transposase-Accessible Chromatin Assay
PBS:: A phosphate buffer saline solution
TSS:: Transcription initiation sites
DMEM:: Dulbecco's Modified Eagle Medium
M:: Methylated
U:: Unmethylated
iNOS:: Inducible nitric oxide synthase
IL1β:: Interleukin 1β
ELISA:: Enzyme-linked immunosorbent assay
MSP:: Methylation-specific PCR
BSAS:: Bisulfite amplicon sequencing
RLU:: Relative light units
EMSA:: Electrophoretic mobility shift assays
SD:: Standard deviation
IHC:: Immunohistochemistry
NCBI:: National Center for Biotechnology Information
TF:: Transcription factor

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Acknowledgements

Our appreciation for the Lv**g Health Service Center's employees is immense, and we are also deeply grateful to the editors, reviewers,and all other people who have aided this research.I greatly appreciate your support and assistance.

Funding

This study was supported in part by grants from the following sources: National Natural Science Foundation of Shandong (Grant No: ZR2022QH332); National Natural Science Foundation of China (Grant No: 8220081418); **an Science and Technology Bureau's Clinical Technology Innovation Program. (Grant No: 202134058); Scientific Research Start-up Funds for talent introduction of **an Central Hospital (Grant No: YJRC2021010).

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Authors and Affiliations

Department of Pulmonary and Critical Care Medicine, Central Hospital Affiliated to Shandong First Medical University, **an, China
Zhen Luo & Shi Zhang
Jiangsu Provincial Key Laboratory of Critical Care Medicine, Department of Critical Care Medicine, School of Medicine, Zhongda Hospital, Southeast University, Nan**g, Jiangsu, China
Zhen Luo & Shi Zhang
Central Hospital Affiliated to Shandong First Medical University, **an, China
Ailing Wang, Wei-sha Zheng, Zhen Luo & Lian Bai

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Ailing Wang
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With complete access to the study's data, SZ took on the responsibility for ensuring its integrity and accuracy. Involvement in the downloading of the data, bioinformatic analysis, and article preparation for publication was also provided by SZ. The manuscript's conclusion has been read and approved by all authors.

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Correspondence to Shi Zhang.

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The Institutional Ethics Committee of Shandong First Medical University's Central Hospital gave its approval and review to this study. The Central Hospital Affiliated to Shandong First Medical University's Animal Care and authorized the evaluation and authorization of animal research conducted in this study. Details regarding the authorization can be found in Additional file 1: Figure S3-S4.

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Supplementary Information

Additional file 1: Table S3.

PCR primers. Table S4. Patients’ basic information. Figure S1. The immunofluorescence staining for validation of CD14⁺ cell. Figure S2. The body weight of the each mice. Figure S3. The Ethics approval and consent for investigation on human CD14⁺ cells. Figure S4. The Ethics approval and consent for animal experiments.

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Wang, A., Zheng, Ws., Luo, Z. et al. The innovative checkpoint inhibitors of lung adenocarcinoma, cg09897064 methylation and ZBP1 expression reduction, have implications for macrophage polarization and tumor growth in lung cancer. J Transl Med 22, 173 (2024). https://doi.org/10.1186/s12967-024-04995-1

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Received: 21 October 2023
Accepted: 12 February 2024
Published: 18 February 2024
DOI: https://doi.org/10.1186/s12967-024-04995-1

The innovative checkpoint inhibitors of lung adenocarcinoma, cg09897064 methylation and ZBP1 expression reduction, have implications for macrophage polarization and tumor growth in lung cancer

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