CCL21/CCR7 interaction promotes cellular migration and invasion via modulation of the MEK/ERK1/2 signaling pathway and correlates with lymphatic metastatic spread and poor prognosis in urinary bladder cancer

Lymph node metastasis in patients with urinary bladder cancer (UBC) is always associated with poor prognosis and is the determinant for tumor staging and the development of treatment regimens; however, its underlying mechanisms remain to be studied. Immunohistochemical staining of tumor sections from 62 UBC patients was performed using CCR7, D2-40 and CD34 antibodies. We showed that increased CCR7 expression was significantly associated with positive lymph node status (P=0.008), pT3-T4 tumor stage (P=0.015), tumor grade (P=0.010) and worse overall survival (OS, P<0.001) and that both CCR7 expression and lymph node metastasis were independent prognostic factors for OS (P=0.031 and P=0.001, respectively) based on multivariate analysis. We found that there was a significant association between MLVD and lymph node status (P=0.006), but this relation was not observed for MVD. Furthermore, we showed that increased CCR7 expression correlated significantly with higher MLVD (P=0.014) and MVD (P=0.002). Wound-healing and Matrigel Transwell assays indicated that activation of CCR7 with CCL21 significantly enhanced the invasion and migration abilities of UM-UC-3 cells, and this enhanced effect was significantly abrogated by CCR7 knockdown using siRNA. Western blot analysis revealed that the phospho-ERK1/2 level was markedly increased when UM-UC-3 cells were treated with CCL21 and significantly decreased when the CCR7 gene was silenced. MEK/ERK1/2 inhibition with PD98059 significantly suppressed the migration and invasion abilities of UM-UC-3 cells and also significantly abrogated the effects of CCL21/CCR7 on cell migration and invasion. Based on these results, we conclude that activation of the CCL21/CCR7 chemoaxis promotes lymph node metastasis of UBC in at least two ways. Firstly, although CCR7 is a promoting factor that induces both lymphangiogenesis and angiogenesis, it may promote lymph node metastasis through its lymphangiogenic effect rather than through its angiogenic effect. Secondly, the CCL21/CCR7 chemoaxis promotes the migration and invasion of UBC cells via the MEK/ERK1/2 signaling pathway rather than the PI3K/AKT pathway.