CXCL14 promotes metastasis of non-small cell lung cancer through ACKR2-depended signaling pathway

Abstract

Background: Lung cancer is a malignant tumor with metastatic potential. Chemokine ligand 14 (CXCL14) has been reported to be associated with different cancer cell migration and invasion. However, few studies have explored the function of CXCL14 and its specific receptor in lung cancer metastasis. This study aims to determine the mechanism of CXCL14-promoted cancer metastasis. Methods: The expression of CXCL14, atypical chemokine receptor 2 (ACKR2), and epithelial mesenchymal transition (EMT) markers was evaluated by the public database of The Cancer Genome Atlas (TCGA) and Gene Expression Omnibus (GEO), Western blot, enzyme-linked immunosorbent assay (ELISA), quantitative real-time polymerase chain reaction (qPCR), immunohistochemistry (IHC), and immunofluorescence (IF). Migration and wound healing assays were used to observe the motility of cancer cells. A luciferase reporter assay was performed to analyze transcription factor activity. The metastasis of lung cancer cells was evaluated in an orthotopic model. Results: We have presented that overexpression of CXCL14 and ACKR2 was observed in lung cancer datasets, human lung tumor sections, and lung cancer cells. Furthermore, the migration of CXCL14-promoted lung cancer cells was determined in vitro and in vivo. In particular, ACKR2 knockdown abolished CXCL14-induced cancer cell motility. Additionally, ACKR2 was involved in CXCL14-triggered phospholipase Cβ3 (PLCβ3), protein kinase Cα (PKCα), and proto-oncogene c-Src signaling pathway and subsequently upregulated nuclear factor κB (NF-κB) transcription activity leading to EMT and migration of lung cancer cells. These results indicated that the CXCL14/ACKR2 axis played an important role in lung cancer metastasis. Conclusion: This study is the first to reveal the function of CXCL14 in promoting EMT and metastasis in lung cancer. As a specific receptor for CXCL14 in lung cancer, ACKR2 mediates CXCL14-induced signaling that leads to cell motility. Our findings can be used as a prognostic biomarker of lung cancer metastasis.

Keywords: ACKR2; CXCL14; epithelial mesenchymal transition; human lung cancer; metastasis.

Figure 1

CXCL14 is overexpressed in lung cancer. (A) Difference in CXCL14 expression between normal and lung adenocarcinoma (LUAD) tissues was analyzed by meta-analysis using data downloaded from Lung Cancer Explorer (LCE). (B-D) The gene expression of CXCL14 in normal lung and LUAD obtained from The Cancer Genome Atlas (TCGA). (E, F) CXCL14 mRNA expression in NSCLC and normal lung tissues in GSE18842 and GSE11969 from the Gene Expression Omnibus (GEO) database. (G) Expression of CXCL14 in normal and tumor lung tissues stained with immunohistochemistry (IHC) and different stages was analyzed using the H score (n = 8). Scale bar = 200 μm. (H) The expression of the CXCL14 protein in MRC-5, H1299, and A549 cells was examined using Western blot (n = 4). (I) CXCL14 secretion was examined in MRC-5, H1299, and A549 cells using an enzyme-linked immunosorbent assay (ELISA; n = 4). Normal tissues and MRC-5 cells were used as controls.

Figure 2

CXCL14 promotes migration and regulates the epithelial mesenchymal transition in lung cancer cells. (A, B) H1299 cells were treated with CXCL14 (1-30 ng/ml) for 24 h, cell migration was measured with migration and wound healing assays (n = 4). (C) H1299 and A549 cells were treated with CXCL14 (1-30 ng/ml) for 24 h, cell viability was evaluated using a CCK-8 assay (n = 4). (D) Expression of CDH2 mRNA in NSCLC and normal lung tissue in GSE18842 from the GEO database. (E) Correlation analysis of CXCL14 and CDH2 in GSE18842. (F) After treatment with CXCL14 (30 ng/ml) for 24 h, the expression levels of E-cadherin, ZO-1, N-cadherin and vimentin were determined in H1299 and A549 cells on immunofluorescence (IF)-stained slides. Scale bar = 20 μm. (G, H) After incubation with CXCL14 (1-30 ng/ml) for 24 h, the expression of epithelial mesenchymal transition (EMT) markers in the H1299 and A549 cells were examined by Western blot (n = 4). Untreated cells were used as controls.

Figure 3

ACKR2 is overexpressed and regulates migration in lung cancer. (A-C) Differences in ACKR2, CXCR4, and GPR85 expression between normal and lung adenocarcinoma (LUAD) tissues were determined through meta-analysis. (D) The expression levels of ACKR2 in immunohistochemistry (IHC)-stained normal and different stage of tumor lung tissues were analyzed using the H score (n = 8). Scale bar = 200 μm. (E) The expression of the ACKR2 protein in MRC-5, H1299 and A549 cells was examined by Western blot. Data are shown as fold of MRC-5 (n = 4). (F, H) H1299 cells were transfected with control siRNA or specific ACKR2, CXCR4, and GPR85 siRNA for 24 h and incubated with CXCL14 (30 ng/ml) for another 24 h (n = 4). Cell migration was assessed through migration and wound healing assays. (G, I) H1299 cells were incubated with IgG or ACKR2, CXCR4, and GPR85 neutralized antibodies (1 μg/ml) for 1 h and incubated with CXCL14 (30 ng/ml) for another 24 h (n = 4). Cell migration was assessed through migration and wound healing assays. Normal tissues, MRC-5 cells, cells transfected with control siRNA, and incubated with IgG were used as controls.

Figure 4

CXCL14 promotes cell migration and EMT marker expression via ACKR2 and PLCβ3, PKCα and c-Src in lung cancer cells. (A) A549 cells were pretreated with U73122 (3 μM), GF109203X (1 μM) or PP2 (1 μM) for 1 h and then treated with CXCL14 (30 ng/ml) for 24 h. Cell migration was measured using a migration assay (n = 4). (B-C) H1299 and A549 cells were treated with CXCL14 (30 ng/ml) and the phosphorylation of PLCβ3, PKCα, and c-Src was examined using Western blot (n = 4). (D) H1299 and A549 cells were pretreated with U73122 or GF109203X for 1 h and then treated with CXCL14 (30 ng/ml) for 15 min. Phosphorylation of c-Src was measured using Western blot (n = 4). (E, F) H1299 cells were pretreated with U73122, GF109203X or PP2 for 1 h and then incubated with CXCL14 (30 ng/ml) for 24 h. Cell migration was measured with migration and wound healing assays (n = 4). (G, H) H1299 and A549 cells were pretreated with U73122, GF109203X or PP2 for 1 h and then treated with CXCL14 (30 ng/ml) for 24 h. The expression of EMT markers was examined using Western blot (n = 4). (I) H1299 and A549 cells were transfected with control siRNA or siRNA specific for PLCβ3, PKCα, or c-Src, and transfection efficiency was measured by Western blot (n = 4). (J, K) H1299 cells were transfected with control siRNA or siRNA specific for PLCβ3, PKCα, or c-Src and then incubated with CXCL14 (30 ng/ml) for 24 h. Cell migration was measured using migration and wound healing assays (n = 4). (L) H1299 and A549 cells were transfected with control siRNA or siRNA specific for ACKR2 siRNA and incubated with CXCL14 (30 ng/ml) for 15 min. The phosphorylation of PLCβ3, PKCα, and c-Src was examined using Western blot (n = 4). Untreated cells and cells transfected with control siRNA were used as controls.

Figure 5

CXCL14 promotes cell migration and EMT marker expression through the IKKα, IκBα, and p65 in lung cancer cells. (A, B) H1299 and A549 cells were treated with CXCL14 (30 ng/ml) to detect the phosphorylation of IKKα, IκBα, and p65 by using Western blot (n = 4). (C) H1299 and A549 cells were pretreated with PP2 for 1 h and treated with CXCL14 (30 ng/ml) for 15 min. The phosphorylation of IKKα was measured using Western blot (n = 4). (D, E) H1299 cells were pretreated with TPCK (1 μM) or BAY11-7082 (0.6 μM) for 1 h and incubated with CXCL14 (30 ng/ml) for 24 h. Cell migration was measured through migration and wound healing assays (n = 4). (F, G) H1299 and A549 cells were pretreated with TPCK or BAY11-7082 for 1 h and incubated with CXCL14 (0 or 30 ng/ml) for 24 h. The expression of EMT markers was examined by Western blot (n = 4). (H) H1299 and A549 cells were transfected with control siRNA or siRNA specific for p65 siRNA, and transfection efficiency was measured using Western blot (n = 4). (I, J) H1299 cells were transfected with siRNA specific for p65 and treated with CXCL14 (30 ng/ml) for 24 h. Cell migration was measured through migration and wound healing assays (n = 4). Untreated cells and cells transfected with control siRNA were used as controls.

Figure 6

CXCL14 promotes NF-κB transcriptional activation via PLCβ3, PKCα, c-Src, IKKα, and IκBα in lung cancer cells. (A, B) H1299 and A549 cells were pretreated with U73122, GF109203X, or PP2 for 1 h and incubated with CXCL14 (30 ng/ml) for 1 h. The phosphorylation of p65 was examined using Western blot (n = 4). (C, D) H1299 and A549 cells were pretreated with TPCK or BAY11-7082 for 1 h and incubated with CXCL14 (30 ng/ml) for 1 h. The phosphorylation of p65 was examined using Western blot (n = 4). (E) H1299 and A549 cells were pretreated with U73122, GF109203X, PP2, TPCK or BAY11-7082 for 1 h and incubated with CXCL14 (30 ng/ml) for 3 h. Cells were stained with anti-p65 antibody and analyzed under an immunofluorescence microscope. The nuclei were counterstained with DAPI. Representative microscopic images are shown. Scale bar = 20 μm. (F) H1299 and A549 cells were transfected with an NF-κB promoter reporter plasmid for 24 h, treated with CXCL14 (1-30 ng/ml) for another 24 h, and evaluated in terms of luciferase activity (n = 4). (G) H1299 and A549 cells were transfected with an NF-κB promoter reporter plasmid for 24 h and pretreated with U73122, GF109203X, PP2, TPCK or BAY11-7082 for 1 h. Cells were incubated with CXCL14 (30 ng/ml) for another 24 h and assessed in terms of luciferase activity (n = 4). Untreated cells were used as controls.

Figure 7

CXCL14 promotes tumor metastasis in the orthotopic model. (A, B) The transfection effects of CXCL14 (OV or KD) in H1299 and A549 cells were examined by Western blot (n = 4). (C, D) The effects of transfection in H1299 and A549 cells were measured using a quantitative real-time polymerase chain reaction (n = 4). (E, F) Cell proliferation of CXCL14-OV and CXCL14-KD cells was examined using a CCK-8 assay (n = 4). (G, H) Cell migration of CXCL14-OV and CXCL14-KD H1299 cells was measured with migration and wound healing assays (n = 4). (I) Schematic illustration of the orthotopic model. (J) Representative images of lung metastases of H1299, CXCL14-OV and CXCL14-KD H1299 vector cells were measured with an in vivo imaging system. The bottom panel showed ex vivo lung tumor images and luminescence intensity of each tumor (n = 4). (K) Representative H&E staining of metastatic lung tumor nodules in each group (n = 4). Scale bar = 4 mm. (L) Representative IHC and H&E staining of orthotopic lung tumor in each group (n = 4). Scale bar = 100 μm.

Figure 8

Schematic mechanism of CXCL14 promotes tumor metastasis in lung cancer. The ligand CXCL14 stimulates the atypical chemokine receptor 2 (ACKR2) to activate PLCβ3, PKCα, and c-Src. The phosphorylation of phospholipase Cβ3 (PLCβ3), protein kinase Cα (PKCα), and proto-oncogene c-Src (c-Src) signaling pathways activates IκB kinase α (IKKα) and NF-κB inhibitor α (IκBα) to release nuclear factor-κB (NF-κB). The nuclear translocation of NF-κB promotes the expression of EMT protein and cell migration that leads to tumor metastasis.