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. 2022 Nov;54(11):1901-1912.
doi: 10.1038/s12276-022-00886-x. Epub 2022 Nov 9.

Particulate matter promotes cancer metastasis through increased HBEGF expression in macrophages

Seung-Ho Park #  1 , Sung-Jin Yoon #  1 , Song Choi #  1   2 , Jaeeun Jung  1 , Jun-Young Park  1 , Young-Ho Park  3 , Jinho Seo  1 , Jungwoon Lee  1   4 , Moo-Seung Lee  1   4 , Seon-Jin Lee  1   4 , Mi-Young Son  4   5 , Young-Lai Cho  6 , Jang-Seong Kim  7 , Hyo Jin Lee  8 , Jinyoung Jeong  9   10 , Dae-Soo Kim  11   12 , Young-Jun Park  13   14
Affiliations

Particulate matter promotes cancer metastasis through increased HBEGF expression in macrophages

Seung-Ho Park et al. Exp Mol Med. 2022 Nov.

Abstract

Although many cohort studies have reported that long-term exposure to particulate matter (PM) can cause lung cancer, the molecular mechanisms underlying the PM-induced increase in cancer metastasis remain unclear. To determine whether PM contributes to cancer metastasis, cancer cells were cultured with conditioned medium from PM-treated THP1 cells, and the migration ability of the treated cancer cells was assessed. The key molecules involved were identified using RNA-seq analysis. In addition, metastatic ability was analyzed in vivo by injection of cancer cells into the tail vein and intratracheal injection of PM into the lungs of C57BL/6 mice. We found that PM enhances the expression of heparin-binding EGF-like growth factor (HBEGF) in macrophages, which induces epithelial-to-mesenchymal transition (EMT) in cancer cells, thereby increasing metastasis. Macrophage stimulation by PM results in activation and subsequent nuclear translocation of the aryl hydrocarbon receptor and upregulation of HBEGF. Secreted HBEGF activates EGFR on the cancer cell surface to induce EMT, resulting in increased migration and invasion in vitro and increased metastasis in vivo. Therefore, our study reveals a critical PM-macrophage-cancer cell signaling axis mediating EMT and metastasis and provides an effective therapeutic approach for PM-induced malignancy.

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Conflict of interest statement

The authors declare no competing interests.

Figures

Fig. 1
Fig. 1. PM exposure changes the characteristics of macrophages.
a Volcano plots of differentially expressed genes in THP1 cells after treatment with the indicated PM types for 24 h. b Multidimensional scaling (MDS) plot of RNA-seq expression profiles in two dimensions. c Heatmap of 452 differentially expressed human cytokine and growth factor genes between THP1-Control cells and THP1-PM2.5, THP1-PM10, and THP1-KRPM cells identified by RNA-seq. d Venn diagram of upregulated cytokines and growth factors. e Venn diagram of downregulated cytokines and growth factors.
Fig. 2
Fig. 2. Conditioned medium (CM) from particulate matter (PM)-treated macrophages increases cancer cell motility and activates EGFR expression in cancer cells.
a Wound healing assay of A549 cells incubated with CM from PM-treated THP1 cells; images were acquired at 0 and 36 h. Scale bar, 250 μm. b Transwell migration (top) and invasion (bottom) assays of A549 cells incubated with CM from PM-treated THP1 cells. Scale bar, 200 μm. c Phospho-RTK array assay of A549 CM-Control (Con) and A549 CM-PM cells. The dots inside the rectangle represent EGFR (left). Relative pixel intensities indicating phospho-EGFR levels in the phospho-RTK array (right). d Gene set enrichment analysis plot of EGFR-induced genes in A549 CM-PM cells. NES normalized enrichment score, FWER familywise error rate, FDR false discovery rate. e Immunoblot analysis of EGFR phosphorylation in A549 cells after incubation with CM from PM-treated THP1 cells for the indicated time. β-Actin was used as the loading control. f Immunoblot analysis of EGFR phosphorylation in A549 cells after incubation with the indicated concentrations of CM from PM-treated THP1 cells at for 20 min. *P ≤ 0.05, ***P ≤ 0.001.
Fig. 3
Fig. 3. Particulate matter (PM)-treated macrophages exhibit increased HBEGF expression.
a Heatmap showing the differential expression of the indicated EGFR ligands between THP1-Control (Con) cells and THP1-PM2.5, THP1-PM10, and THP1-KRPM cells identified by RNA-seq. b qRT‒PCR analysis of THP1 cells treated with the indicated concentrations of different types of PM for 24 h. c ELISA of THP1 cells treated with the indicated concentrations of different types of PM for 24 h. AD Arizona dust. d Immunoblot analysis of THP1 cells treated with the indicated concentrations of different types of PM for 24 h. e qRT‒PCR analysis of human cord blood-derived macrophages treated with 5 μg/cm2 PM for 24 h. f ELISA of human cord blood-derived macrophages treated with 5 μg/cm2 PM for 24 h. *P ≤ 0.05, **P ≤ 0.01, ***P ≤ 0.001.
Fig. 4
Fig. 4. HBEGF secreted by particulate matter (PM)-treated macrophages increases cancer cell motility and activates EGFR in cancer cells.
a ELISA of conditioned medium from particulate matter-treated cells (CM-PM) immunoprecipitated with control IgG or an anti-HBEGF antibody. b Immunoblot analysis of EGFR phosphorylation in A549 cells after treatment with CM-PM immunoprecipitated with control IgG or an anti-HBEGF antibody. c Wound healing assay of A549 cells treated with CM-PM immunoprecipitated using control IgG or an anti-HBEGF antibody. Images were acquired at 0 and 36 h. Scale bar, 250 μm. d Transwell migration (top) and invasion (bottom) assays of A549 cells treated with CM-PM immunoprecipitated using control IgG or an anti-HBEGF antibody. Scale bar, 200 μm. *P ≤ 0.05, **P ≤ 0.01, ***P ≤ 0.001; ns not significant.
Fig. 5
Fig. 5. Conditioned medium (CM) from particulate matter (PM)-treated macrophages induces epithelial-to-mesenchymal transition (EMT) in cancer cells.
a Heatmap of differential gene expression between A549 CM-Control (Con) and A549 CM-PM cells identified by RNA-seq. b Gene set enrichment analysis plot for the “Epithelial-to-mesenchymal transition” gene set from the HALLMARK gene collection. NES normalized enrichment score; FWER familywise error rate; FDR false discovery rate. c Immunoblot analysis of A549 cells incubated for 24 h with the indicated concentration of CM from PM-treated THP1 cells. β-Actin was used as the loading control. d Immunoblot analysis of A549 cells treated with CM-PM immunoprecipitated using control IgG or an anti-HBEGF antibody for 24 h. e qRT‒PCR analysis of A549 cells incubated for 24 h with the indicated concentration of CM from PM-treated THP1 cells. f qRT‒PCR analysis of A549 cells treated with CM-PM immunoprecipitated using control IgG or an anti-HBEGF antibody for 24 h. g Immunofluorescence analysis of A549 cells cultured with CM-PM immunoprecipitated using control IgG or an anti-HBEGF antibody for 24 h. Scale bar, 20 μm. *P ≤ 0.05, **P ≤ 0.01, ***P ≤ 0.001; ns not significant.
Fig. 6
Fig. 6. Activation of the aryl hydrocarbon receptor (AhR) by particulate matter (PM) in macrophages increases HBEGF expression.
a Heatmap of the differential expression of AhR-related genes between THP1-Control (Con) cells and THP1-PM2.5, THP1-PM10, and THP1-KRPM cells identified by RNA-seq. b qRT‒PCR analysis of THP1 cells treated with different types of PM (25 μg/cm2) for 24 h. c Immunoblot analysis of PM2.5 (25 μg/cm2)-stimulated AhR translocation. β-Actin and Lamin A were used as the cytoplasmic fraction and nuclear fraction loading controls, respectively. d Immunoblot analysis of THP1 cells treated with the indicated concentrations of PM2.5 for 24 h. e, f qRT-PCR (e) and ELISA (f) of THP1 cells transfected with 50 nM siAhR and treated with 25 μg/cm2 PM2.5 for 24 h. g, h qRT‒PCR (g) and ELISA (h) of THP1 cells pretreated with 10 nM CH-223191 for 1 h and treated with 25 μg/cm2 PM2.5 for an additional 24 h. *P ≤ 0.05, **P ≤ 0.01, ***P ≤ 0.001; ns not significant.
Fig. 7
Fig. 7. The increase in the HBEGF level induced by particulate matter (PM) contributes to lung cancer metastasis in mice.
a Hematoxylin and eosin (H&E) staining of lung samples harvested from control and PM-treated mice. Black arrow, immune cells; white arrow, PM. Scale bar, 100 μm. b Quantification of total cells in the bronchoalveolar lavage fluid (BALF) of control and PM-treated mice. c qRT‒PCR analysis of whole lungs from control and PM-treated mice. d qRT‒PCR analysis of macrophages from the lungs of control and PM-treated mice. e Immunoblot analysis of BALF (top line), plasma (middle line), and whole-lung lysate (bottom line) from control and PM-treated mice. β-Actin was used as the loading control for the whole-lung lysate. f In vivo metastasis assay with intravenously injected luciferase-expressing Lewis lung carcinoma (LLC-luc) cells; 24 h after intravenous injection of cells, mice were injected intratracheally three times over 3 days with PM alone or with CRM197 (an inhibitor of HBEGF). IVIS images were acquired 14 days after intravenous injection of cells. g Quantification of bioluminescence (n = 5 mice per group). h Representative images of H&E staining from the in vivo metastasis assay. Scale bar, 500 μm. i Quantification of metastatic lung nodules. j Proposed model of metastasis with PM-exposed macrophages. PM induces HBEGF expression in macrophages through activation of NF-κB and AP-1, which promotes EMT in cancer cells and facilitates metastasis. *P ≤ 0.05, **P ≤ 0.01, ***P ≤ 0.001; ns not significant.
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