Jak3 is involved in CCR7-dependent migration and invasion in metastatic squamous cell carcinoma of the head and neck

Abstract

Patients with cervical lymph node metastasis in squamous cell carcinoma of the head and neck (SCCHN) exhibit a poor prognosis and low 5-year survival rate. It has been proven that chemokine receptor 7 (CCR7) promotes cellular migration and invasion in metastatic SCCHN. In the present study, the metastatic SCCHN PCI-37B cell line was utilized to explore the role of Janus activated kinase-3 (Jak3) in the CCR7-mediated signaling pathway in metastatic SCCHN cells. It was observed that phospho-Jak3 was expressed in SCCHN tissues. In addition, when the PCI-37B cells were analyzed in response to chemokine ligand 19 (CCL19), the ligand of CCR7, at the indicated time points, the results of the present study demonstrated that CCR7 induced Jak3 activation, and inhibition of Jak3 activity using a specific inhibitor, ZM39923, significantly attenuated CCR7-induced Jak3 phosphorylation. Migration and invasion assays and immunofluorescence staining experiments demonstrated that CCL19 promoted cell migration, invasion and F-actin rearrangment in CCR7-expressing SCCHN cells partially due to the activation of the Jak3 signaling pathway. These results demonstrate that the Jak3 signaling pathway is important for the CCR7-induced malignant biological behavior of SCCHN cells.

Keywords: Janus activated kinase-3; chemokine receptor 7; squamous cell carcinoma of the head and neck.

Figure 1.

Expression of phospho-Jak3 was analyzed by immunohistochemistry. Representative stained images of normal oral mucosal tissues, SCCHN tissue and metastatic lymph nodes. Scale bars, 200 µm. Jak3, Janus activated kinase-3; SCCHN, squamous cell carcinoma of the head and neck.

Figure 2.

Jak3 phosphorylation induced by CCL19 in CCR7-expressing SCCHN cells was examined by western blot assay. (A) Time-course effect of CCL19 on Jak3 phosphorylation examined in PCI-37B cells. PCI-37B cells of control and CCL19 groups were treated with vehicle or 200 ng/ml CCL19 for 0, 0.25, 1 5, 10, 15 and 30 min, respectively. (B) Effect of CCR7 Ab and ZM39923 on CCL19-induced Jak3 activation in PCI-37B cells. PCI-37B cells pretreated with CCR7 Ab or ZM39923 followed by CCL19 were stimulated with 200 ng/ml CCL19 for 5 min. Jak3, Janus activated kinase-3; CCL19, chemokine ligand 19; CCR7, chemokine receptor 7; SCCHN, squamous cell carcinoma of the head and neck; Ab, antibody.

Figure 3.

Jak3 activation enhances metastatic potential of SCCHN cells. (A) The cell migration rate was compared by wound healing assay. Wound closure was followed at 0 and 24 h subsequent to scratching the cell layer. (B) ZM39923 attenuated the cell migration and invasion induced by CCL19 application. Representative images and corresponding statistical data. Scale bars, 100 µm. Data are presented as the mean ± standard deviation (n=3, each group). *P<0.05 vs. the control group, **P<0.05 vs. group of CCL19 alone. SCCHN, squamous cell carcinoma of the head and neck; CCL19, chemokine ligand 19; CCR7, chemokine receptor 7.

Figure 4.

Jak3 inhibition blocks reorganization of the actin cytoskeleton induced by CCL19 in PCI-37B cells. The cells were stained with rhodamine-labeled phalloidin. Representative immunostaining images exhibiting control, CCL19 alone, CCR7 Ab followed by CCL19 and ZM39923 followed by CCL19 (original magnification, ×400). Similar results were obtained from 3 independent experiments. Jak3, Janus activated kinase-3; CCL19, chemokine ligand 19; CCR7, chemokine receptor 7; Ab, antibody.