Research Article: CCL21/CCR7 Promotes G2/M Phase Progression via the ERK Pathway in Human Non-Small Cell Lung Cancer Cells

Date Published: June 16, 2011

Publisher: Public Library of Science

Author(s): Ying Xu, Lifeng Liu, Xueshan Qiu, Lili Jiang, Bo Huang, Haiying Li, Zixuan Li, Wenting Luo, Enhua Wang, Lin Zhang.

Abstract: C-C chemokine receptor 7 (CCR7) contributes to the survival of certain cancer cell lines, but its role in the proliferation of human non-small cell lung cancer (NSCLC) cells remains vague. Proliferation assays performed on A549 and H460 NSCLC cells using Cell Counting Kit-8 indicated that activation of CCR7 by its specific ligand, exogenous chemokine ligand 21 (CCL21), was associated with a significant linear increase in cell proliferation with duration of exposure to CCL21. The CCL21/CCR7 interaction significantly increased the fraction of cells in the G2/M phase of the cell cycle as measured by flow cytometry. In contrast, CCL21/CCR7 had no significant influence on the G0/G1 and S phases. Western blot and real-time PCR indicated that CCL21/CCR7 significantly upregulated expression of cyclin A, cyclin B1, and cyclin-dependent kinase 1 (CDK1), which are related to the G2/M phase transition. The expression of cyclin D1 and cyclin E, which are related to the G0/G1 and G1/S transitions, was not altered. The CCL21/CCR7 interaction significantly enhanced phosphorylation of extracellular signal-regulated kinase (P-ERK) but not Akt, as measured by Western blot. LY294002, a selective inhibitor of PI3K that prevents activation of the downstream Akt, did not weaken the effect of CCL21/CCR7 on P-ERK. Coimmunoprecipitation further confirmed that there was an interaction between P-ERK and cyclin A, cyclin B1, or CDK1, particularly in the presence of CCL21. CCR7 small interfering RNA or PD98059, a selective inhibitor of MEK that disrupts the activation of downstream ERK, significantly abolished the effects of exogenous CCL21. These results suggest that CCL21/CCR7 contributes to the time-dependent proliferation of human NSCLC cells by upregulating cyclin A, cyclin B1, and CDK1 potentially via the ERK pathway.

Partial Text: C-C chemokine receptor 7 (CCR7) is expressed on all naive T-cells and on some memory T-cells, B-cells, and mature dendritic cells [1]. Upon interaction with its ligands, chemokine ligand 19 (CCL19) or chemokine ligand 21 (CCL21) [2], CCR7 contributes to lymphocyte trafficking and homing to lymph nodes during immune and inflammatory reactions [3]–[5]. CCR7 is highly expressed in non-small cell lung cancer (NSCLC), breast cancer, and squamous cell carcinoma of the head and neck and is responsible for mediating metastasis in certain cancer cells lines [6]–[15]. To date, the role of CCR7 in the proliferation of human NSCLC cells has not been elucidated.

CCL21/CCR7 promotes proliferation of A549 and H460 cells. In a previous study, we identified a higher CCR7 expression level in A549 and H460 human NSCLC cell lines compared with other cell lines [32]. To investigate the role of CCR7 in the functioning of A549 and H460 cells, CCR7 activation and inhibition were induced with exogenous CCL21 and with CCR7 small interfering RNA (siRNA), respectively. After transfection with CCR7 siRNA (siCCR7) or control siRNA, the expression of CCR7 was evaluated using Western blot and reverse transcriptase (RT)-PCR. We found that siCCR7 significantly downregulated the protein and mRNA levels of CCR7, compared with control siRNA (Figure 1).

Several studies have documented that the activation of CCR7 is responsible for mediating survival of certain cancer cell lines by promoting migration and proliferation or by inhibiting apoptosis [33], [35]–[42]. However, the role of CCR7 in the proliferation of human NSCLC cells has not been well documented. In the present study, we confirmed that the CCL21/CCR7 interaction can significantly enhance human NSCLC cell proliferation in a time-dependent manner, involving the upregulation of cyclin A, cyclin B1, and CDK1, possibly via the ERK, but not the Akt, pathway.