Chemokine (C‑C motif) ligand 21/C‑C chemokine receptor type 7 triggers migration and invasion of human lung cancer cells by epithelial‑mesenchymal transition via the extracellular signal‑regulated kinase signaling pathway

Abstract

C-C chemokine receptor type 7 (CCR7) has been implicated in lymph node metastasis of various cancers. Previous studies have revealed that epithelial‑mesenchymal transition (EMT) is involved in the chemotactic process mediated by CCR7 and its ligands in various types of carcinoma. However, the underlying mechanism of this process remains to be fully elucidated. The present study investigated whether chemokine (C‑C motif) ligand 21 (CCL21)/CCR7 may activate EMT of lung cancer cells and their associated signaling pathways. A549 and H520 lung cancer cell lines were examined in vitro in the present study. The results indicated that A549 and H520 expressed CCR7, but reduced levels of CCL21. Following stimulation of lung cancer cell lines with CCL21, the expression of the epithelial marker E‑cadherin was downregulated, and the mesenchymal markers Vimentin/Slug and extracellular signal‑regulated kinase (ERK) were upregulated. In addition, the ERK inhibitor PD98059 may inhibit EMT caused by CCL21, and decreased cell migration and invasion initiated by CCL21. Furthermore, lung adenocarcinoma tissues from 50 patients who underwent lung cancer operations were investigated by immunohistochemistry. The findings revealed that CCR7, Slug and Vimentin were highly expressed in lung carcinoma tissues, and were significantly associated with lymph node metastasis and clinical pathological stages, respectively. CCR7 expression was correlated positively with expression levels of Slug and Vimentin. CCL21 was expressed positively in the endothelium of lymphatic vessels adjacent to cancer cells, and weakly in lung cancer cells. Collectively, these results demonstrated that CCL21/CCR7 may activate EMT in lung cancer cells via the ERK1/2 signaling pathway. The current study provides evidence that a close interaction exists between CCL21/CCR7chemotaxis and EMT procedures in lung cancer metastasis, providing a basis for the development of therapeutic targets.

Figure 1.

Detection of CCR7, CCL21, EMT biomarkers and p-ERK expression in response to CCL21 in lung cancer cells. Representative western blot images of (A) CCR7 (B) E-cadherin, Vimentin, Slug and (C) p-ERK protein expression levels. β-actin served as an internal loading control. CCR7, C-C chemokine receptor type 7; CCL21, chemokine (C-C motif) ligand 21; EMT, epithelial-mesenchymal transition; ERK, extracellular-regulated signal kinase; p, phosphorylated.

Figure 2.

Roles of CCL21/CCR7 and p-ERK in EMT of lung cancer cells. (A) Western blot and (B) reverse transcription-quantitative polymerase chain reaction. Cancer cells presented a downregulation of E-cadherin and upregulation of Vimentin, Slug and p-ERK in response to CCL21 stimulation, when compared with the control group. These phenotype transformations in response to CCL21 stimulation above were abolished when the cells were pretreated with PD98059. Data are presented as the mean ± standard deviation of three independent experiments. **P<0.01. CCL21, chemokine (C-C motif) ligand 21; CCR7, C-C chemokine receptor type 7; ERK, extracellular-regulated signal kinase; EMT, epithelial-mesenchymal transition.

Figure 3.

Effect of ERK pathway in CCL21/CCR7 axis on migratory capacity of human lung cancer cells. (A) H520 and (B) A549 cells were subjected to wound-healing assay. (C) Quantification of the wound healing assay; more cancer cells migrated towards to wound area in response to CCL21 stimulation, when compared with the control group, whereas this was inhibited when the cells were pretreated with PD98059. Magnification, ×200. Scale bar=200 µm. **P<0.01. ERK, extracellular-regulated signal kinase; CCL21, chemokine (C-C motif) ligand 21; CCR7, C-C chemokine receptor type 7.

Figure 4.

Evaluation of invasive capacity in human lung cancer cells following ERK pathway inhibition. (A) H520 and (B) A549 cells were subjected to a Matrigel invasive assay. (C) The number of invasive cells treated with CCL21 was increased significantly in H520 and A549 cells, whereas this was significantly inhibited when the cells were pretreated with PD98059. Cells were imaged under a phase-contrast microscope. Magnification, ×200. Scale bar=50 µm. **P<0.01. ERK, extracellular-regulated signal kinase; CCL21, chemokine (C-C motif) ligand 21.

Figure 5.

Immunohistochemical detection of CCR7, Slug, Vimentin and CCL21 expression in human lung cancer tissues. (A) CCR7 was strongly stained in the cytoplasm and/or plasma membrane of human lung cancer cells. (B) Slug was stained primarily in the nucleus and partly in the cytoplasm of cancer cells. (C) Vimentin were strongly stained in the cytoplasm and/or plasma membrane. (D) CCL21 was stained mainly in the lymphatic vessels around the cancer foci (as indicated by the black arrow). Negative control staining for (E) CCR7, (F) Slug, (G) Vimentin and (H) CCL21. Magnification, ×200. Scale bar=200 µm. CCR7, C-C chemokine receptor type 7; CCL21, chemokine (C-C motif) ligand 21.