CCL21-CCR7 promotes the lymph node metastasis of esophageal squamous cell carcinoma by up-regulating MUC1

Abstract

Background: CCR7 and MUC1 are correlated with lymph node metastasis in ESCC, but the role of MUC1 in the CCR7-induced lymphatic metastasis and the underlying molecular mechanism is still unclear.

Methods: The expression of CCR7 and MUC1 was detected in the ESCC samples by IHC, and the clinical significance of CCR7 and MUC1 in ESCC was analyzed. The expression of CCR7 and MUC1 in ESCC cell lines was detected by qRT-PCR and western blot. The effect of CCL21 on the migration and invasion of ESCC cells was determined by transwell assay. The activity of MUC1 promoter was determined by luciferase reporter assay. The activation of Erk, Akt and Sp1 was detected by western blot and the binding of Sp1 to the MUC1 promoter was determined by ChIP.

Results: The co-expression of CCR7 and MUC1 was detected in 153 ESCC samples by IHC, and both were correlated with lymph node metastasis, regional lymphatic recurrence and poor prognosis. Correspondingly, increasing levels of MUC1 mRNA and protein were detected in the ESCC cell lines KYSE410 and Eca9706 after treatment with CCL21 in a time- and dose-dependent manner. Furthermore, silencing MUC1 could remarkably suppress the invasion and migration of ESCC cells induced by CCL21. Moreover, heterologous CCR7 promoted the invasion and migration of KYSE150 and up-regulated MUC1 expression. Increasing levels of activated ERK1/2 and Akt were detected in KYSE410 after treating the cells with CCL21, and inhibiting the activation of ERK1/2 but not Akt caused the increased transcription of MUC1. Finally, the phosphorylation of Sp1 induced by ERK1/2 and subsequent increases in the binding of Sp1 to the muc1 promoter at -99/-90 were confirmed to cause the up-regulation of MUC1 induced by CCL21-CCR7.

Conclusions: Our findings suggested that MUC1 plays an important role in CCL21-CCR7-induced lymphatic metastasis and may serve as a therapeutic target in ESCC.

Fig. 1

Co-expression of CCR7 and MUC1 in ESCC sample. a The expression of CCR7 and MUC1 in ESCC detected by IHC; b the immunoreactivity score of MUC1 in group with CCR7 positive expression and CCR7 negative expression group; c the 3-year regional recurrence curve of patients with CCR7 and MUC1 positive/negative expression; d the 5-year survival curve of patients with CCR7 and MUC1 positive/negative expression

Fig. 2

CCL21 induced the up-regulation of MUC1 in ESCC cell lines. a, b the mRNA and protein expression level of MUC1 and CCR7 in ESCC cell lines; c: CCL21 induced the increasing MUC1 mRNA in ESCC cells. KYSE150, KYSE410, KYSE450 and Eca9706 were starved for 24 h and then treated with CCL21 at a concentration of 100 ng/ml for 12 h, then cells were harvested for the qRT-PCR; d, e CCL21 up regulated mRNA of MUC1 in a time and dose dependent way. Eca9706 and KYSE410 were starved for 24 h and then treated with CCL21 at a concentration of 0, 25, 50, 100, 200 ng/ml for 12 h or cells were treated with CCL21 at a concentration of 100 ng/ml for 0, 2, 6, 12 or 24 h. Then cells were harvested for qRT-PCR; f, g increasing of MUC1-C in KYSE410 and Eca9706 after treated with CCL21 at concentration of 0, 25,50, 100, 200 ng/ml for 24 h confirmed by immunoblotting; h, f blocking CCR7suppressed the up-regulation of MUC1 induced by CCL21. Eca9706 and KYSE410 were pretreated by CCR7 antibody (1 ug/mL) or IgG as control, then treated by 100 ng/ml for 24 h, then cells were harvested for detection of MUC1 by immunoblotting; j expression of MUC1 in KYSE410 and Eca9706 treated with PBS or CCL21(100 ng/mL) detected by immunofluorescence. Each data point represents the mean ± SD of three repeated experiments. *P < 0.05

Fig. 3

Silencing of MUC1 suppressed migration and invasion induced by CCL21. Cells were starved for 24 h and then seeding into the upper chamber, for the migration assay CCL21 was added into the lower chamber at a concentration of 200 ng/ml and incubated for 12 h; for the invasion assay the CCL21 was added into the upper chamber at a concentration of 200 ng/ml and incubated for 36 h. Cells on the lower surface of the membrane were counted in five randomly selected fields. a CCL21 promoted migration and invasion of KYSE410 and Eca9706 while blocking CCR7 could reverse migration and invasion induced by CCL21, and silencing MUC1 by siRNA targeted to MUC1 significantly suppressed the migration and invasion induced by CCL21; b Total cell numbers on the lower surface of the membrane counted in five randomly selected fields. Each data point represents the mean ± SD of 3 repeated experiments. *P < 0.05; Each datapoint represents the mean ± SD of three repeated experiments. *P < 0.05

Fig. 4

Activation of ERK1/2 was responsible for the up-regulation of MUC1 induced by CCL21-CCR7. a The activation of ERK1/2 and Akt pathway induced by CCL21, KYSE410 and Eca9706 cells were seeding into 6 well culture plate and starved overnight, then culture medium was replaced by serum free medium contained CCL21 (100 ng/ml) and incubated for 0, 15, 30, 45, 60 min. Then cells were harvested for immunoblotting; b Blocking CCR7 could suppress the activation of Akt and ERK1/2 pathway induced by CCL21 in KYSE410 and Eca9706; c, d Inhibiting activation of ERK1/2 but not Akt could suppress the up-regulation of MUC1-C protein. The starved KYSE410 and Eca9706 cells were pretreated by DMSO as controll, U0126 or MK2206 for 30 min, then cells were treated with PBS or CCL21 (100 ng/ml). For detecting p-ERK1/2, p-Akt and MUC1-C, cells were harvested after incubated with CCL21 for 15 min, 30 min and 24 h respectively; e Inhibiting ERK1/2 but not Akt could remarkably suppress the activity of MUC1 promoter, KYSE410 and Eca9706 cells transfected with MUC1-pGL2b plasmid were pretreated with DMSO as control, U0126 or MK2206 and then treated with PBS or CCL21 (100 ng/ml) for 12 h, then cells were harvested to detect the relative luciferase activity. Each datapoint represents the mean ± SD of three repeated experiments. *P < 0.05

Fig. 5

Phosphorylation of SP1 was responsible for the up-regulation of MUC1 induced by CCL21-CCR7. a, b Silencing Sp1 could remarkably suppressed the up-regulation of MUC1 in KYSE410 and Eca9706 induced by CCL21. The starved siSp1-Eca9706, siNC-Eca9706, siSp1-KYSE410 and siNC-KYSE410 were treated with CCL21(100 ng/mL) for 24 h, then cells were harvested for immunoblotting for MUC1-C; c Silencing Sp1/Mutant of Sp1 binding site at −99/−97 could remarkably suppressed MUC1 promoter activity induced by CCL21. siSp1-Eca9706, siNC-Eca9706, siSp1-KYSE410 and siNC-KYSE410 transfected by MUC1-pGL2b luciferase reporter plasmid/KYSE410 and Eca9706 cells transfected by MUC1-pGL2b or MUC1 mutant-pGL2b -firefly luciferase reporter plasmid were treated with PBS or CCL21(100 ng/mL) for 24 h, then cells were harvested for detecting the luciferase activity; d Increasing phosphorylation of Sp1 induced by CCL21. Starved KYSE410 and Eca9706 cells were treated with CCL21(100 ng/mL) for 0, 0.5, 1,2 and 6 h, then cells were harvested for the immunoblot of p-Sp1; e The expression of p-Sp1 in KYSE410 treated with PBS or CCL21(100 ng/ml) for 6 h detected by immunofluorescence; f Blocking CCR7 could suppress the phosphorylation of Sp1 induced by CCL21. The starved KYSE410 and Eca9706 cells pretreated with the CCR7 antibody or IgG as control, were treated with PBS or CCL21 for 6 h, then cells were harvested for the immunoblot of p-Sp1; g Inhibiting ERK1/2 suppressed phosphorylation of SP1 induced by CCL21. Starved KYSE410 and Eca9706 cells were pretreated with DMSO as control or U0126 for 30 min. After treated with PBS or CCL21(100 ng/mL) for 6 h, the cells were harvested for immunoblot; h Inhibiting ERK1/2 suppressed Sp1 binding to MUC1 promoter at −99/−97. Starved KYSE410 cells were pretreated with DMSO as control or U0126 for 0.5. After treated with PBS or CCL21(100 ng/mL) for 12 h, the cells were harvested for the ChIP assay. The targeted DNA was amplified using MUC1 primers with 40 cycles of PCR. Each datapoint represents the mean ± SD of three repeated experiments. *P < 0.05

Fig. 6

Heterologous CCR7 promoted migration and invasion and up-regulated expression of MUC1 in KYSE150. a Heterologous expression of CCR7 up regulated the expression of MUC1 mRNA in KYSE150, KYSE150-CCR7 and KYSE150NC cells were starved and then treated with PBS or CCL21(0, 25, 50, 100, 200 ng/mL) for 12 h and then harvested for qRT-PCR and the result showed the remarkable up-regulation of MUC1 in KYSE150-CCR7 after treated with CCL21 compared to the KYSE150NC groups; b Heterologous expression of CCR7 up regulated the expression of MUC1-C protein in KYSE150, KYSE150-CCR7 and KYSE150NC cells were starved and then treated with PBS or CCL21(100 ng/mL) for 24 h and then harvested for immunoblot and the result showed the remarkable up-regulation of MUC1 in KYSE150-CCR7 after treated with CCL21 compared to the KYSE150NC groups; c Heterologous expression of CCR7 promoted migration and invasion induced by CCL21, the starved KYSE150-CCR7 and KYSE150NC cells were seeding into the upper chamber, for the migration assay CCL21 was added into the lower chamber at a concentration of 200 ng/ml and incubated for 12 h; for the invasion assay the CCL21 was added into the upper chamber and incubated for 36 h; d Total cell numbers on the lower surface of the membrane counted in five randomly selected fields. Each data point represents the mean ± SD of three repeated experiments. *P < 0.05